Compositions and methods for producing isoprene free of c5 hydrocarbons under decoupling conditions and/or safe operating ranges

ABSTRACT

The invention features methods for producing isoprene from cultured cells wherein the cells in the stationary phase. The invention also provides compositions that include these cultured cells and/or increased amount of isoprene. The invention also provides for systems that include a non-flammable concentration of isoprene in the gas phase. Additionally, the invention provides isoprene compositions, such as compositions with increased amount of isoprene or increased purity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/496,573, filed Jul. 1, 2009, which claims the priority benefit ofU.S. Provisional Application No. 61/134,094, filed Jul. 2, 2008, U.S.Provisional Application No. 61/133,947, filed Jul. 2, 2008, and U.S.Provisional Application No. 61/134,011, filed Jul. 2, 2008, the contentsof each are hereby incoroporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Isoprene (2-methyl-1,3-butadiene) is the critical starting material fora variety of synthetic polymers, most notably synthetic rubbers.Isoprene is naturally produced by a variety of microbial, plant, andanimal species. In particular, two pathways have been identified for thebiosynthesis of isoprene: the mevalonate (MVA) pathway and thenon-mevalonate (DXP) pathway (FIG. 19). However, the yield of isoprenefrom naturally-occurring organisms is commercially unattractive. About800,000 tons per year of cis-polyisoprene are produced from thepolymerization of isoprene; most of this polyisoprene is used in thetire and rubber industry. Isoprene is also copolymerized for use as asynthetic elastomer in other products such as footwear, mechanicalproducts, medical products, sporting goods, and latex.

Currently, the tire and rubber industry is based on the use of naturaland synthetic rubber. Natural rubber is obtained from the milky juice ofrubber trees or plants found in the rainforests of Africa. Syntheticrubber is based primarily on butadiene polymers. For these polymers,butadiene is obtained as a co-product from ethylene and propylenemanufacture.

While isoprene can be obtained by fractionating petroleum, thepurification of this material is expensive and time-consuming. Petroleumcracking of the C5 stream of hydrocarbons produces only about 15%isoprene. Thus, more economical methods for producing isoprene areneeded. In particular, methods that produce isoprene at rates, titers,and purity that are sufficient to meet the demands of a robustcommercial process are desirable. Also desired are systems for producingisoprene from inexpensive starting materials.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention features cells in culture that produceisoprene. In some embodiments, the invention provides cells in culturethat produce greater than about 400 nmole of isoprene/gram of cells forthe wet weight of the cells/hour (nmole/g_(wcm)/hr) of isoprene. In someembodiments, the cells have a heterologous nucleic acid that (i) encodesan isoprene synthase polypeptide and (ii) is operably linked to apromoter. In some embodiments, the cells are cultured in a culturemedium that includes a carbon source, such as, but not limited to, acarbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source,oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, polypeptide (e.g., a microbial or plant protein or peptide),yeast extract, component from a yeast extract, or any combination of twoor more of the foregoing. In some embodiments, the cells are culturedunder limited glucose conditions.

In some embodiments, the invention provides cells in culture thatconvert more than about 0.002% of the carbon in a cell culture mediuminto isoprene. In some embodiments, the cells have a heterologousnucleic acid that (i) encodes an isoprene synthase polypeptide and (ii)is operably linked to a promoter. In some embodiments, the cells arecultured in a culture medium that includes a carbon source, such as, butnot limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone,one-carbon source, oil, animal fat, animal oil, fatty acid, lipid,phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride,renewable carbon source, polypeptide (e.g., a microbial or plant proteinor peptide), yeast extract, component from a yeast extract, or anycombination of two or more of the foregoing. In some embodiments, thecells are cultured under limited glucose conditions.

In some embodiments, the invention provides cells in culture thatcomprise a heterologous nucleic acid encoding an isoprene synthasepolypeptide. In some embodiments, the cells have a heterologous nucleicacid that (i) encodes an isoprene synthase polypeptide and (ii) isoperably linked to a promoter. In some embodiments, the cells arecultured in a culture medium that includes a carbon source, such as, butnot limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone,one-carbon source, oil, animal fat, animal oil, fatty acid, lipid,phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride,renewable carbon source, polypeptide (e.g., a microbial or plant proteinor peptide), yeast extract, component from a yeast extract, or anycombination of two or more of the foregoing. In some embodiments, thecells are cultured under limited glucose conditions.

In one aspect, the invention features methods of producing isoprene,such as methods of using any of the cells described herein to produceisoprene. In some embodiments, the method involves culturing cells underconditions sufficient to produce greater than about 400 nmole/g_(wcm)/hrof isoprene. In some embodiments, the method also includes recoveringisoprene produced by the cells. In some embodiments, the method includespurifying isoprene produced by the cells. In some embodiments, themethod includes polymerizing the isoprene. In some embodiments, thecells have a heterologous nucleic acid that (i) encodes an isoprenesynthase polypeptide and (ii) is operably linked to a promoter. In someembodiments, the cells are cultured in a culture medium that includes acarbon source, such as, but not limited to, a carbohydrate, glycerol,glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animaloil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride,diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., amicrobial or plant protein or peptide), yeast extract, component from ayeast extract, or any combination of two or more of the foregoing. Insome embodiments, the cells are cultured under limited glucoseconditions. In various embodiments, the amount of isoprene produced(such as the total amount of isoprene produced or the amount of isopreneproduced per liter of broth per hour per OD₆₀₀) during stationary phaseis greater than or about 2 or more times the amount of isoprene producedduring the growth phase for the same length of time. In someembodiments, the gas phase comprises greater than or about 9.5% (volume)oxygen, and the concentration of isoprene in the gas phase is less thanthe lower flammability limit or greater than the upper flammabilitylimit. In particular embodiments, (i) the concentration of isoprene inthe gas phase is less than the lower flammability limit or greater thanthe upper flammability limit, and (ii) the cells produce greater thanabout 400 nmole/g_(wcm)/hr of isoprene.

In some embodiments, the method includes culturing cells underconditions sufficient to convert more than about 0.002% of the carbon(mol/mol) in a cell culture medium into isoprene. In some embodiments,the method also includes recovering isoprene produced by the cells. Insome embodiments, the method includes purifying isoprene produced by thecells. In some embodiments, the method includes polymerizing theisoprene. In some embodiments, the cells have a heterologous nucleicacid that (i) encodes an isoprene synthase polypeptide and (ii) isoperably linked to a promoter. In some embodiments, the cells arecultured in a culture medium that includes a carbon source, such as, butnot limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone,one-carbon source, oil, animal fat, animal oil, fatty acid, lipid,phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride,renewable carbon source, polypeptide (e.g., a microbial or plant proteinor peptide), yeast extract, component from a yeast extract, or anycombination of two or more of the foregoing. In some embodiments, thecells are cultured under limited glucose conditions.

In some embodiments, isoprene is only produced in stationary phase. Insome embodiments, isoprene is produced in both the growth phase andstationary phase. In various embodiments, the amount of isopreneproduced (such as the total amount of isoprene produced or the amount ofisoprene produced per liter of broth per hour per OD₆₀₀) duringstationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40,50, or more times the amount of isoprene produced during the growthphase for the same length of time.

In one aspect, the invention features compositions and systems thatcomprise isoprene. In some embodiments, the composition comprisesgreater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg of isoprene. In someembodiments, the composition comprises greater than or about 2, 5, 10,20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene (w/w) of the volatileorganic fraction of the composition is isoprene.

In some embodiments, the composition comprises greater than or about99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight comparedto the total weight of all C5 hydrocarbons in the composition. In someembodiments, the composition comprises less than or about 0.12, 0.10,0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or0.00001% C5 hydrocarbons other than isoprene (such 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1-pentyne, 2-pentyne,1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, trans-piperylene,cis-piperylene, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne) by weight compared to the total weight of all C5hydrocarbons in the composition. In some embodiments, the compositionhas less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005,0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1-pentyne, 2-pentyne,1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, trans-piperylene,cis-piperylene, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne by weight compared to the total weight of all C5hydrocarbons in the composition. In particular embodiments, thecomposition has greater than about 2 mg of isoprene and has greater thanor about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weightcompared to the total weight of all C5 hydrocarbons in the composition.

In some embodiments, the composition has less than or about 50, 40, 30,20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ug/L of a compound thatinhibits the polymerization of isoprene for any compound in thecomposition that inhibits the polymerization of isoprene. In particularembodiments, the composition also has greater than about 2 mg ofisoprene.

In some embodiments, the composition has one or more compounds selectedfrom the group consisting of ethanol, acetone, C5 prenyl alcohols, andisoprenoid compounds with 10 or more carbon atoms. In some embodiments,the composition has greater than or about 0.005, 0.01, 0.05, 0.1, 0.5,1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 ug/L of ethanol, acetone, a C5prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol),or any two or more of the foregoing. In particular embodiments, thecomposition has greater than about 2 mg of isoprene and has one or morecompounds selected from the group consisting of ethanol, acetone, C5prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms.

In some embodiments, the composition includes isoprene and one or moresecond compounds selected from the group consisting of 2-heptanone,6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine,2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methylacetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethylacetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone,1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate,3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate,3-methyl-3-but-1-enyl acetate, 3-methyl-2-but-1-enyl acetate,(E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene,and 2,3-cycloheptenolpyridine. In various embodiments, the amount of oneof these second components relative to the amount of isoprene in unitsof percentage by weight (i.e., weight of the component divided by theweight of isoprene times 100) is at greater than or about 0.01, 0.02,0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110%(w/w).

In some embodiments, the composition comprises (i) a gas phase thatcomprises isoprene and (ii) cells in culture that produce greater thanabout 400 nmole/g_(wcm)/hr of isoprene. In some embodiments, thecomposition comprises a closed system, and the gas phase comprisesgreater than or about 5. 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ug/L ofisoprene when normalized to 1 mL of 1 OD₆₀₀ cultured for 1 hour. In someembodiments, the composition comprises an open system, and the gas phasecomprises greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100 ug/L of isoprene when sparged at a rate of 1 vvm. In someembodiments, the volatile organic fraction of the gas phase comprisesgreater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100%isoprene by weight compared to the total weight of all C5 hydrocarbonsin the volatile organic fraction. In some embodiments, the volatileorganic fraction of the gas phase comprises less than or about 0.12,0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001,0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,trans-piperylene, cis-piperylene, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared tothe total weight of all C5 hydrocarbons in the volatile organicfraction. In some embodiments, the volatile organic fraction of the gasphase has less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01,0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,trans-piperylene, cis-piperylene, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne by weight compared tothe total weight of all C5 hydrocarbons in the volatile organicfraction. In particular embodiments, the volatile organic fraction ofthe gas phase has greater than about 2 mg of isoprene and has greaterthan or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene byweight compared to the total weight of all C5 hydrocarbons in thevolatile organic fraction.

In some embodiments, the volatile organic fraction of the gas phase hasless than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or0.005 ug/L of a compound that inhibits the polymerization of isoprenefor any compound in the volatile organic fraction of the gas phase thatinhibits the polymerization of isoprene. In particular embodiments, thevolatile organic fraction of the gas phase also has greater than about 2mg of isoprene.

In some embodiments, the volatile organic fraction of the gas phase hasone or more compounds selected from the group consisting of ethanol,acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or morecarbon atoms. In some embodiments, the volatile organic fraction of thegas phase has greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5,10, 20, 30, 40, 60, 80, 100, or 120 ug/L of ethanol, acetone, a C5prenyl alcohol (such as 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol),or any two or more of the foregoing. In particular embodiments, thevolatile organic fraction of the gas phase has greater than about 2 mgof isoprene and has one or more compounds selected from the groupconsisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoidcompounds with 10 or more carbon atoms.

In some embodiments, the volatile organic fraction of the gas phase hasincludes isoprene and one or more second compounds selected from thegroup consisting of 2-heptanone, 6-methyl-5-hepten-2-one,2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal,acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl,2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol,3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone,3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butylacetate, 3-methylbutyl acetate, 3-methyl-3-but-1-enyl acetate,3-methyl-2-but-1-enyl acetate, (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine. Invarious embodiments, the amount of one of these second componentsrelative to amount of isoprene in units of percentage by weight (i.e.,weight of the component divided by the weight of isoprene times 100) isat greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, or 110% (w/w) in the volatile organicfraction of the gas phase.

In some embodiments of any of the compositions of the invention, atleast a portion of the isoprene is in a gas phase. In some embodiments,at least a portion of the isoprene is in a liquid phase (such as acondensate). In some embodiments, at least a portion of the isoprene isin a solid phase. In some embodiments, at least a portion of theisoprene is adsorbed to a solid support, such as a support that includessilica and/or activated carbon. In some embodiments, the compositionincludes ethanol. In some embodiments, the composition includes betweenabout 75 to about 90% by weight of ethanol, such as between about 75 toabout 80%, about 80 to about 85%, or about 85 to about 90% by weight ofethanol. In some embodiments, the composition includes between about 4to about 15% by weight of isoprene, such as between about 4 to about 8%,about 8 to about 12%, or about 12 to about 15% by weight of isoprene.

In some embodiments, the invention also features systems that includeany of the cells and/or compositions described herein. In someembodiments, the system includes a reactor that chamber comprises cellsin culture that produce greater than about 400, 500, 600, 700, 800, 900,1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or morenmole/g_(wcm)/hr isoprene. In some embodiments, the system is not aclosed system. In some embodiments, at least a portion of the isopreneis removed from the system. In some embodiments, the system includes agas phase comprising isoprene. In various embodiments, the gas phasecomprises any of the compositions described herein.

In one aspect, the invention provides a tire comprising polyisoprene. Insome embodiments, the polyisoprene is produced by (i) polymerizingisoprene in any of the compositions described herein or (ii)polymerizing isoprene recovered from any of the compositions describedherein. In some embodiments, the polyisoprene comprisescis-1,4-polyisoprene.

In some embodiments of any of the compositions, systems, and methods ofthe invention, a nonflammable concentration of isoprene in the gas phaseis produced. In some embodiments, the gas phase comprises less thanabout 9.5% (volume) oxygen. In some embodiments, the gas phase comprisesgreater than or about 9.5% (volume) oxygen, and the concentration ofisoprene in the gas phase is less than the lower flammability limit orgreater than the upper flammability limit. In some embodiments, theportion of the gas phase other than isoprene comprises between about 0%to about 100% (volume) oxygen, such as between about 10% to about 100%(volume) oxygen. In some embodiments, the portion of the gas phase otherthan isoprene comprises between about 0% to about 99% (volume) nitrogen.In some embodiments, the portion of the gas phase other than isoprenecomprises between about 1% to about 50% (volume) CO₂.

In some embodiments of any of the aspects of the invention, the cells inculture produce isoprene at greater than or about 400, 500, 600, 700,800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000,or more nmole/g_(wcm)/hr isoprene. In some embodiments of any of theaspects of the invention, the cells in culture convert greater than orabout 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6%, or more of the carbonin the cell culture medium into isoprene. In some embodiments of any ofthe aspects of the invention, the cells in culture produce isoprene atgreater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500,600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000,4,000, 5,000, 10,000, 100,000, or more ng of isoprene/gram of cells forthe wet weight of the cells/hr (ng/g_(wcm)/h). In some embodiments ofany of the aspects of the invention, the cells in culture produce acumulative titer (total amount) of isoprene at greater than or about 1,10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900,1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000,50,000, 100,000, or more mg of isoprene/L of broth (mg/L_(broth),wherein the volume of broth includes the volume of the cells and thecell medium). Other exemplary rates of isoprene production and totalamounts of isoprene production are disclosed herein.

In some embodiments of any of the aspects of the invention, the cellsfurther comprise a heterologous nucleic acid encoding an IDIpolypeptide. In some embodiments of any of the aspects of the invention,the cells further comprise an insertion of a copy of an endogenousnucleic acid encoding an IDI polypeptide. In some embodiments of any ofthe aspects of the invention, the cells further comprise a heterologousnucleic acid encoding a DXS polypeptide. In some embodiments of any ofthe aspects of the invention, the cells further comprise an insertion ofa copy of an endogenous nucleic acid encoding a DXS polypeptide. In someembodiments of any of the aspects of the invention, the cells furthercomprise one or more nucleic acids encoding an IDI polypeptide and a DXSpolypeptide. In some embodiments of any of the aspects of the invention,one nucleic acid encodes the isoprene synthase polypeptide, IDIpolypeptide, and DXS polypeptide. In some embodiments of any of theaspects of the invention, one vector encodes the isoprene synthasepolypeptide, IDI polypeptide, and DXS polypeptide. In some embodiments,the vector comprises a selective marker, such as an antibioticresistance nucleic acid.

In some embodiments of any of the aspects of the invention, theheterologous isoprene synthase nucleic acid is operably linked to a T7promoter, such as a T7 promoter contained in a medium or high copyplasmid. In some embodiments of any of the aspects of the invention, theheterologous isoprene synthase nucleic acid is operably linked to a Trcpromoter, such as a Trc promoter contained in a medium or high copyplasmid. In some embodiments of any of the aspects of the invention, theheterologous isoprene synthase nucleic acid is operably linked to a Lacpromoter, such as a Lac promoter contained in a low copy plasmid. Insome embodiments of any of the aspects of the invention, theheterologous isoprene synthase nucleic acid is operably linked to anendogenous promoter, such as an endogenous alkaline serine proteasepromoter. In some embodiments, the heterologous isoprene synthasenucleic acid integrates into a chromosome of the cells without aselective marker.

In some embodiments, one or more MVA pathway, IDI, DXP, or isoprenesynthase nucleic acids are placed under the control of a promoter orfactor that is more active in stationary phase than in the growth phase.For example, one or more MVA pathway, IDI, DXP, or isoprene synthasenucleic acids may be placed under control of a stationary phase sigmafactor, such as RpoS. In some embodiments, one or more MVA pathway, IDI,DXP, or isoprene synthase nucleic acids are placed under control of apromoter inducible in stationary phase, such as a promoter inducible bya response regulator active in stationary phase.

In some embodiments of any of the aspects of the invention, at least aportion of the cells maintain the heterologous isoprene synthase nucleicacid for at least or about 5, 10, 20, 40, 50, 60, 65, or more celldivisions in a continuous culture (such as a continuous culture withoutdilution). In some embodiments of any of the aspects of the invention,the nucleic acid comprising the isoprene synthase, IDI, or DXS nucleicacid also comprises a selective marker, such as an antibiotic resistancenucleic acid.

In some embodiments of any of the aspects of the invention, the cellsfurther comprise a heterologous nucleic acid encoding an MVA pathwaypolypeptide (such as an MVA pathway polypeptide from Saccharomycescerevisia or Enterococcus faecalis). In some embodiments of any of theaspects of the invention, the cells further comprise an insertion of acopy of an endogenous nucleic acid encoding an MVA pathway polypeptide(such as an MVA pathway polypeptide from Saccharomyces cerevisia orEnterococcus faecalis). In some embodiments of any of the aspects of theinvention, the cells comprise an isoprene synthase, DXS, and MVA pathwaynucleic acid. In some embodiments of any of the aspects of theinvention, the cells comprise an isoprene synthase nucleic acid, a DXSnucleic acid, an IDI nucleic acid, and a MVA pathway nucleic (inaddition to the IDI nucleic acid).

In some embodiments of any of the aspects of the invention, the isoprenesynthase polypeptide is a naturally-occurring polypeptide from a plantsuch as Pueraria (e.g., Pueraria montana or Pueraria lobata).

In some embodiments of any of the aspects of the invention, the cellsare bacterial cells, such as gram-positive bacterial cells (e.g.,Bacillus cells such as Bacillus subtilis cells or Streptomyces cellssuch as Streptomyces lividans, Streptomyces coelicolor, or Streptomycesgriseus cells). In some embodiments of any of the aspects of theinvention, the cells are gram-negative bacterial cells (e.g.,Escherichia cells such as Escherichia coli cells or Pantoea cells suchas Pantoea citrea cells). In some embodiments of any of the aspects ofthe invention, the cells are fungal, cells such as filamentous fungalcells (e.g., Trichoderma cells such as Trichoderma reesei cells orAspergillus cells such as Aspergillus oryzae and Aspergillus niger) oryeast cells (e.g., Yarrowia cells such as Yarrowia lipolytica cells).

In some embodiments of any of the aspects of the invention, themicrobial polypeptide carbon source includes one or more polypeptidesfrom yeast or bacteria. In some embodiments of any of the aspects of theinvention, the plant polypeptide carbon source includes one or morepolypeptides from soy, corn, canola, jatropha, palm, peanut, sunflower,coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower,sesame, or linseed.

In one aspect, the invention features a product produced by any of thecompositions or methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleotide sequence of a kudzu isoprene synthase genecodon-optimized for expression in E. coli (SEQ ID NO:1). The atg startcodon is in italics, the stop codon is in bold and the added PstI siteis underlined.

FIG. 2 is a map of pTrcKudzu.

FIG. 3A-3C is the nucleotide sequence of pTrcKudzu (SEQ ID NO:2). TheRBS is underlined, the kudzu isoprene synthase start codon is in boldcapitol letters and the stop codon is in bold, capitol, italics letters.The vector backbone is pTrcHis2B.

FIG. 4 is a map of pETNHisKudzu.

FIG. 5A-5C is the nucleotide sequence of pETNHisKudzu (SEQ ID NO:5).

FIG. 6 is a map of pCL-lac-Kudzu.

FIG. 7A-7C is the nucleotide sequence of pCL-lac-Kudzu (SEQ ID NO:7).

FIG. 8A is a graph showing the production of isoprene in E. coli BL21cells with no vector.

FIG. 8B is a graph showing the production of isoprene in E. coli BL21cells with pCL-lac-Kudzu

FIG. 8C is a graph showing the production of isoprene in E. coli BL21cells with pTrcKudzu.

FIG. 8D is a graph showing the production of isoprene in E. coli BL21cells with pETN-H isKudzu.

FIG. 9A is a graph showing OD over time of fermentation of E. coliBL21/pTrcKudzu in a 14 liter fed batch fermentation.

FIG. 9B is a graph showing isoprene production over time of fermentationof E. coli BL21/pTrcKudzu in a 14 liter fed batch fermentation.

FIG. 10A is a graph showing the production of isoprene in Panteoacitrea. Control cells without recombinant kudzu isoprene synthase. Greydiamonds represent isoprene synthesis, black squares represent OD₆₀₀).

FIG. 10B is a graph showing the production of isoprene in Panteoa citreaexpressing pCL-lac Kudzu. Grey diamonds represent isoprene synthesis,black squares represent OD₆₀₀.

FIG. 10C is a graph showing the production of isoprene in Panteoa citreaexpressing pTrcKudzu. Grey diamonds represent isoprene synthesis, blacksquares represent OD₆₀₀).

FIG. 11 is a graph showing the production of isoprene in Bacillussubtilis expressing recombinant isoprene synthase. BG3594comK is a B.subtilis strain without plasmid (native isoprene production).CF443-BG3594comK is a B. subtilis strain with pBSKudzu (recombinantisoprene production). IS on the y-axis indicates isoprene.

FIG. 12A-12C is the nucleotide sequence of pBS Kudzu #2 (SEQ ID NO:57).

FIG. 13 is the nucleotide sequence of kudzu isoprene synthasecodon-optimized for expression in Yarrowia (SEQ ID NO:8).

FIG. 14 is a map of pTrex3 g comprising a kudzu isoprene synthase genecodon-optimized for expression in Yarrowia.

FIG. 15A-15C is the nucleotide sequence of vector pSPZ1(MAP29Spb) (SEQID NO:11).

FIG. 16 is the nucleotide sequence of the synthetic kudzu (Puerariamontana) isoprene gene codon-optimized for expression in Yarrowia (SEQID NO:12).

FIG. 17 is the nucleotide sequence of the synthetic hybrid poplar(Populus alba×Populus tremula) isoprene synthase gene (SEQ ID NO:13).The ATG start codon is in bold and the stop codon is underlined.

FIGS. 18A1 and 18A2 shows a schematic outlining construction of vectorspYLA 1, pYL1 and pYL2 (SEQ ID NO:79, SEQ ID NO:73-77).

FIG. 18B shows a schematic outlining construction of the vectorpYLA(POP1) (SEQ ID NO:70-73).

FIG. 18C shows a schematic outlining construction of the vectorpYLA(KZ1)

FIG. 18D shows a schematic outlining construction of the vectorpYLI(KZ1) (SEQ IDS NOs:103-104).

FIG. 18E shows a schematic outlining construction of the vectorpYLI(MAP29)

FIG. 18F shows a schematic outlining construction of the vectorpYLA(MAP29)

FIG. 19 shows the MVA and DXP metabolic pathways for isoprene (based onF. Bouvier et al., Progress in Lipid Res. 44: 357-429, 2005). Thefollowing description includes alternative names for each polypeptide inthe pathways and a reference that discloses an assay for measuring theactivity of the indicated polypeptide (each of these references are eachhereby incorporated by reference in their entireties, particularly withrespect to assays for polypeptide activity for polypeptides in the MVAand DXP pathways). Mevalonate Pathway: AACT; Acetyl-CoAacetyltransferase, MvaE, EC 2.3.1.9. Assay: J. Bacteriol., 184:2116-2122, 2002; HMGS; Hydroxymethylglutaryl-CoA synthase, MvaS, EC2.3.3.10. Assay: J. Bacteriol., 184: 4065-4070, 2002; HMGR;3-Hydroxy-3-methylglutaryl-CoA reductase, MvaE, EC 1.1.1.34. Assay: J.Bacteriol., 184: 2116-2122, 2002; MVK; Mevalonate kinase, ERG12, EC2.7.1.36. Assay: Curr Genet. 19:9-14, 1991. PMK; Phosphomevalonatekinase, ERGS, EC 2.7.4.2, Assay: Mol Cell Biol., 11:620-631, 1991;DPMDC; Diphosphomevalonate decarboxylase, MVD1, EC 4.1.1.33. Assay:Biochemistry, 33:13355-13362, 1994; IDI; Isopentenyl-diphosphatedelta-isomerase, IDI1, EC 5.3.3.2. Assay: J. Biol. Chem.264:19169-19175, 1989. DXP Pathway: DXS; 1-Deoxyxylulose-5-phosphatesynthase, dxs, EC 2.2.1.7. Assay: PNAS, 94:12857-62, 1997; DXR;1-Deoxy-D-xylulose 5-phosphate reductoisomerase, dxr, EC 2.2.1.7. Assay:Eur. J. Biochem. 269:4446-4457, 2002; MCT;4-Diphosphocytidyl-2C-methyl-D-erythritol synthase, IspD, EC 2.7.7.60.Assay: PNAS, 97: 6451-6456, 2000; CMK;4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase, IspE, EC 2.7.1.148.Assay: PNAS, 97:1062-1067, 2000; MCS; 2C-Methyl-D-erythritol2,4-cyclodiphosphate synthase, IspF, EC 4.6.1.12. Assay: PNAS,96:11758-11763, 1999; HDS; 1-Hydroxy-2-methyl-2-(E)-butenyl4-diphosphate synthase, ispG, EC 1.17.4.3. Assay: J. Org. Chem.,70:9168-9174, 2005; HDR; 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphatereductase, IspH, EC 1.17.1.2. Assay: JACS, 126:12847-12855, 2004.

FIG. 20 shows graphs representing results of the GC-MS analysis ofisoprene production by recombinant Y. lipolytica strains without (left)or with (right) a kudzu isoprene synthase gene. The arrows indicate theelution time of the authentic isoprene standard.

FIG. 21 is a map of pTrcKudzu yIDI DXS Kan.

FIG. 22A-22D is the nucleotide sequence of pTrcKudzu yIDI DXS Kan (SEQID NO:20).

FIG. 23A is a graph showing production of isoprene from glucose inBL21/pTrcKudzukan. Time 0 is the time of induction with IPTG (400 μmol).The x-axis is time after induction; the y-axis is OD₆₀₀ and the y2-axisis total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23B is a graph showing production of isoprene from glucose inBL21/pTrcKudzu yIDI kan. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23C is a graph showing production of isoprene from glucose inBL21/pTrcKudzu DXS kan. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23D is a graph showing production of isoprene from glucose inBL21/pTrcKudzu yIDI DXS kan. Time 0 is the time of induction with IPTG(400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ andthe y2-axis is total productivity of isoprene (μg/L headspace orspecific productivity (μg/L headspace/OD). Diamonds represent OD₆₀₀,circles represent total isoprene productivity (μg/L) and squaresrepresent specific productivity of isoprene (μg/L/OD).

FIG. 23E is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23F is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu yIDI. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23G is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu DXS. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23H is a graph showing production of isoprene from glucose inBL21/pTrcKudzuIDIDXSkan. The arrow indicates the time of induction withIPTG (400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀and the y2-axis is total productivity of isoprene (μg/L headspace orspecific productivity (μg/L headspace/OD). Black diamonds representOD₆₀₀, black triangles represent isoprene productivity (μg/L) and whitesquares represent specific productivity of isoprene (μg/L/OD).

FIG. 24 is a map of pTrcKKDyIkIS kan.

FIG. 25A-25D is a nucleotide sequence of pTrcKKDyIkIS kan (SEQ IDNO:33).

FIG. 26 is a map of pCL PtrcUpperPathway.

FIGS. 27A-27D is a nucleotide sequence of pCL PtrcUpperPathway (SEQ IDNO:46).

FIG. 28 shows a map of the cassette containing the lower MVA pathway andyeast idi for integration into the B. subtilis chromosome at the nprElocus. nprE upstream/downstream indicates 1 kb each of sequence from thenprE locus for integration. aprE promoter (alkaline serine proteasepromoter) indicates the promoter (−35, −10, +1 transcription start site,RBS) of the aprE gene. MVK1 indicates the yeast mevalonate kinase gene.RBS-PMK indicates the yeast phosphomevalonte kinase gene with a BacillusRBS upstream of the start site. RBS-MPD indicates the yeastdiphosphomevalonate decarboxylase gene with a Bacillus RBS upstream ofthe start site. RBS-IDI indicates the yeast idi gene with a Bacillus RBSupstream of the start site. Terminator indicates the terminator alkalineserine protease transcription terminator from B. amyliquefaciens. SpecRindicates the spectinomycin resistance marker. “nprE upstream repeat foramp.” indicates a direct repeat of the upstream region used foramplification.

FIG. 29A-29D is a nucleotide sequence of cassette containing the lowerMVA pathway and yeast idi for integration into the B. subtilischromosome at the nprE locus (SEQ ID NO:47).

FIG. 30 is a map of p9796-poplar.

FIG. 31A-31B is a nucleotide sequence of p9796-poplar (SEQ ID NO:48).

FIG. 32 is a map of pTrcPoplar.

FIG. 33A-33C is a nucleotide sequence of pTrcPoplar (SEQ ID NO:49).

FIG. 34 is a map of pTrcKudzu yIDI Kan.

FIG. 35A-35C is a nucleotide sequence of pTrcKudzu yIDI Kan (SEQ IDNO:50).

FIG. 36 is a map of pTrcKudzuDXS Kan.

FIG. 37A-37C is a nucleotide sequence of pTrcKudzuDXS Kan (SEQ IDNO:51).

FIG. 38 is a map of pCL PtrcKudzu.

FIG. 39A-39C is a nucleotide sequence of pCL PtrcKudzu (SEQ ID NO:52).

FIG. 40 is a map of pCL PtrcKudzu A3.

FIG. 41A-41C is a nucleotide sequence of pCL PtrcKudzu A3 (SEQ IDNO:53).

FIG. 42 is a map of pCL PtrcKudzu yIDI.

FIG. 43A-43C is a nucleotide sequence of pCL PtrcKudzu yIDI (SEQ IDNO:54).

FIG. 44 is a map of pCL PtrcKudzu DXS.

FIG. 45A-45D is a nucleotide sequence of pCL PtrcKudzu DXS (SEQ IDNO:55).

FIG. 46A-46E shows graphs representing isoprene production from biomassfeedstocks. Panel A shows isoprene production from corn stover, Panel Bshows isoprene production from bagasse, Panel C shows isopreneproduction from softwood pulp, Panel D shows isoprene production fromglucose, and Panel E shows isoprene production from cells with noadditional feedstock. Grey squares represent OD₆₀₀ measurements of thecultures at the indicated times post-inoculation and black trianglesrepresent isoprene production at the indicated times post-inoculation.

FIG. 47A shows a graph representing isoprene production by BL21 (λDE3)pTrcKudzu yIDI DXS (kan) in a culture with no glucose added. Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47B shows a graph representing isoprene production from 1% glucosefeedstock invert sugar by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47C shows a graph representing isoprene production from 1% invertsugar feedstock by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 47D shows a graph representing isoprene production from 1% AFEXcorn stover feedstock by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squaresrepresent OD₆₀₀, and triangles represent isoprene produced (μg/ml).

FIG. 48A-48C shows graphs demonstrating the effect of yeast extract ofisoprene production. Panel A shows the time course of optical densitywithin fermentors fed with varying amounts of yeast extract. Panel Bshows the time course of isoprene titer within fermentors fed withvarying amounts of yeast extract. The titer is defined as the amount ofisoprene produced per liter of fermentation broth. Panel C shows theeffect of yeast extract on isoprene production in E. coli grown infed-batch culture.

FIG. 49A-49C shows graphs demonstrating isoprene production from a 500 Lbioreactor with E. coli cells containing the pTrcKudzu+yIDI+DXS plasmid.Panel A shows the time course of optical density within the 500-Lbioreactor fed with glucose and yeast extract. Panel B shows the timecourse of isoprene titer within the 500-L bioreactor fed with glucoseand yeast extract. The titer is defined as the amount of isopreneproduced per liter of fermentation broth. Panel C shows the time courseof total isoprene produced from the 500-L bioreactor fed with glucoseand yeast extract.

FIG. 50 is a map of pJMupperpathway2.

FIG. 51A-51C is the nucleotide sequence of pJMupperpathway2 (SEQ IDNO:56).

FIG. 52 is a map of pBS Kudzu #2.

FIG. 53A is a graph showing growth during fermentation time of Bacillusexpressing recombinant kudzu isoprene synthase in 14 liter fed batchfermentation. Black diamonds represent a control strain (BG3594comK)without recombinant isoprene synthase (native isoprene production) andgrey triangles represent Bacillus with pBSKudzu (recombinant isopreneproduction).

FIG. 53B is a graph showing isoprene production during fermentation timeof Bacillus expressing recombinant kudzu isoprene synthase in 14 literfed batch fermentation. Black diamonds represent a control strain(BG3594comK) without recombinant isoprene synthase (native isopreneproduction) and grey triangles represent Bacillus with pBSKudzu(recombinant isoprene production).

FIG. 54 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 55 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 56 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 57 is a time course of optical density within the 15-L bioreactorfed with glycerol.

FIG. 58 is a time course of isoprene titer within the 15-L bioreactorfed with glycerol. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 59 is a time course of total isoprene produced from the 15-Lbioreactor fed with glycerol.

FIGS. 60A-60C are the time courses of optical density, mevalonic acidtiter, and specific productivity within the 150-L bioreactor fed withglucose.

FIGS. 61A-61C are the time courses of optical density, mevalonic acidtiter, and specific productivity within the 15-L bioreactor fed withglucose.

FIGS. 62A-62C are the time courses of optical density, mevalonic acidtiter, and specific productivity within the 15-L bioreactor fed withglucose.

FIG. 63A-63C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 64A-64C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 65A-65C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 66A-66C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIG. 67A-67C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIG. 68 is a graph of the calculated adiabatic flame temperatures forSeries A as a function of fuel concentration for various oxygen levels.The figure legend lists the curves in the order in which they appear inthe graph. For example, the first entry in the figure legend (isoprenein air at 40° C.) corresponds to the highest curve in the graph.

FIG. 69 is a graph of the calculated adiabatic flame temperatures forSeries B as a function of fuel concentration for various oxygen levelswith 4% water. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 70 is a graph of the calculated adiabatic flame temperatures forSeries C as a function of fuel concentration for various oxygen levelswith 5% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 71 is a graph of the calculated adiabatic flame temperatures forSeries D as a function of fuel concentration for various oxygen levelswith 10% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 72 is a graph of the calculated adiabatic flame temperatures forSeries E as a function of fuel concentration for various oxygen levelswith 15% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 73 is a graph of the calculated adiabatic flame temperatures forSeries F as a function of fuel concentration for various oxygen levelswith 20% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 74 is a graph of the calculated adiabatic flame temperatures forSeries G as a function of fuel concentration for various oxygen levelswith 30% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 75A is a table of the conversion of the CAFT Model results fromweight percent to volume percent for series A.

FIG. 75B is a graph of the flammability results from the CAFT model forSeries A in FIG. 68 plotted as volume percent.

FIG. 76A is a table of the conversion of the CAFT Model results fromweight percent to volume percent for series B.

FIG. 76B is a graph of the flammability results from the CAFT model forSeries B in FIG. 69 plotted as volume percent.

FIG. 77 is a figure of the flammability test vessel.

FIG. 78A is a graph of the flammability Curve for Test Series 1: 0%Steam, 0 psig, and 40° C.

FIG. 78B is a table summarizing the explosion and non-explosion datapoints for Test Series 1.

FIG. 78C is a graph of the flammability curve for Test Series 1 comparedwith the CAFT Model.

FIG. 79A is a graph of the flammability curve for Test Series 2: 4%Steam, 0 psig, and 40° C.

FIG. 79B is a table summarizing the explosion and non-explosion datapoints for Test Series 2.

FIG. 79C is a graph of the flammability curve for Test Series 2 comparedwith the CAFT Model.

FIGS. 80A and 80B are a table of the detailed experimental conditionsand results for Test Series 1.

FIG. 81 is a table of the detailed experimental conditions and resultsfor Test Series 2.

FIG. 82 is a graph of the calculated adiabatic flame temperature plottedas a function of fuel concentration for various nitrogen/oxygen ratiosat 3 atmospheres of pressure.

FIG. 83 is a graph of the calculated adiabatic flame temperature plottedas a function of fuel concentration for various nitrogen/oxygen ratiosat 1 atmosphere of pressure.

FIG. 84 is a graph of the flammability envelope constructed using datafrom FIG. 82 and following the methodology described in Example 13. Theexperimental data points (circles) are from tests described herein thatwere conducted at 1 atmosphere initial system pressure.

FIG. 85 is a graph of the flammability envelope constructed using datafrom FIG. 83 and following the methodology described in Example 13. Theexperimental data points (circles) are from tests described herein thatwere conducted at 1 atmosphere initial system pressure.

FIG. 86A is a GC/MS chromatogram of fermentation off-gas.

FIG. 86B is an expansion of FIG. 86A to show minor volatiles present infermentation off-gas.

FIG. 87A is a GC/MS chromatogram of trace volatiles present in off-gasfollowing cryo-trapping at −78° C.

FIG. 87B is a GC/MS chromatogram of trace volatiles present in off-gasfollowing cryo-trapping at −196° C.

FIG. 87C is an expansion of FIG. 87B.

FIG. 87D is an expansion of FIG. 87C.

FIGS. 88A and 88B are GC/MS chromatogram comparing C5 hydrocarbons frompetroleum-derived isoprene (FIG. 88A) and biologically produced isoprene(FIG. 88B). The standard contains three C5 hydrocarbon impuritieseluting around the main isoprene peak (FIG. 88A). In contrast,biologically produced isoprene contains amounts of ethanol and acetone(run time of 3.41 minutes) (FIG. 88A).

FIG. 89 is a graph of the analysis of fermentation off-gas of an E. coliBL21 (DE3) pTrcIS strain expressing a Kudzu isoprene synthase and fedglucose with 3 g/L yeast extract.

FIG. 90 shows the structures of several impurities that are structurallysimilar to isoprene and may also act as polymerization catalyst poisons.

FIG. 91 is a map of pTrcHis2AUpperPathway (also called pTrcUpperMVA).

FIGS. 92A-92C are the nucleotide sequence of pTrcHis2AUpperPathway (alsocalled pTrcUpperMVA) (SEQ ID NO:86).

FIG. 93 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 94 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 95 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 96 is a time course of optical density within the 15-L bioreactorfed with invert sugar.

FIG. 97 is a time course of isoprene titer within the 15-L bioreactorfed with invert sugar. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 98 is a time course of total isoprene produced from the 15-Lbioreactor fed with invert sugar.

FIG. 99 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 100 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 101 is a time course of isoprene specific activity from the 15-Lbioreactor fed with glucose.

FIG. 102 is a map of pCLPtrcUpperPathwayHGS2.

FIGS. 103A-103C are the nucleotide sequence of pCLPtrcUpperPathwayHGS2(SEQ ID NO:87).

FIG. 104 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 105 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 106 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 107 is a map of plasmid MCM330.

FIGS. 108A-108C are the nucleotide sequence of plasmid MCM330 (SEQ IDNO:90).

FIG. 109 is a map of pET24D-Kudzu.

FIGS. 110A and 110B are the nucleotide sequence of pET24D-Kudzu (SEQ IDNO:101).

FIG. 111A is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 111B is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 111C is a time course of specific productivity of isoprene in the15-L bioreactor fed with glucose.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention features compositions and methods for theproduction of isoprene in increased amounts and/or purity. As usedherein, the term “isoprene” or “2-methyl-1,3-butadiene” (CAS#78-79-5)refers to the direct and final volatile C5 hydrocarbon product from theelimination of pyrophosphate from 3,3-dimethylallyl pyrophosphate(DMAPP), and does not involve the linking or polymerization of one ormore isopentenyl diphosphate (IPP) molecules to one or more DMAPPmolecules.

The vast majority of isoprene is derived from petrochemical sources asan impure C5 hydrocarbon fraction which requires extensive purificationbefore the material is suitable for polymerization. Several impuritiesare particularly problematic given their structural similarity toisoprene and the fact that they can act as polymerization catalystpoisons. Such compounds include 1,3-cyclopentadiene, cis- andtrans-1,3-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne,trans-piperylene, cis-piperylene, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne (FIG. 90). In someembodiments, the isoprene composition of the invention is substantiallyfree of any contaminating unsaturated C5 hydrocarbons. As describedfurther in Example 10, no detectable amount of unsaturated C5hydrocarbons other than isoprene (such as 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1-pentyne, 2-pentyne,1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, trans-piperylene,cis-piperylene, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne) was found in isoprene compositions produced usingthe methods described herein. Some isoprene compositions produced usingthe methods described herein contain ethanol, acetone, and C5 prenylalcohols as determined by GC/MS analysis. All of these components arefar more readily removed from the isoprene stream than the isomeric C5hydrocarbon fractions that are present in isoprene compositions derivedfrom petrochemical sources. Accordingly, in some embodiments, theisoprene compositions of the invention require minimal treatment inorder to be of polymerization grade.

In one aspect, compositions and methods of the invention increase therate of isoprene production and increase the total amount of isoprenethat is produced. For example, cell culture systems that generate4.8×10⁴ nmole/g_(wcm)/hr of isoprene have been produced (Table 1). Theefficiency of these systems is demonstrated by the conversion of about2.2% of the carbon that the cells consume from a cell culture mediuminto isoprene. As shown in the Examples and Table 2, approximately 3 gof isoprene per liter of broth was generated. If desired, even greateramounts of isoprene can be obtained using other conditions, such asthose described herein. In some embodiments, a renewable carbon sourceis used for the production of isoprene. In some embodiments, theproduction of isoprene is decoupled from the growth of the cells. Insome embodiments, the concentrations of isoprene and any oxidants arewithin the nonflammable ranges to reduce or eliminate the risk that afire may occur during production or recovery of isoprene. Thecompositions and methods of the present invention are desirable becausethey allow high isoprene yield per cell, high carbon yield, highisoprene purity, high productivity, low energy usage, low productioncost and investment, and minimal side reactions. This efficient, largescale, biosynthetic process for isoprene production provides an isoprenesource for synthetic isoprene-based rubber and provides a desirable,low-cost alternative to using natural rubber.

As discussed further below, the amount of isoprene produced by cells canbe greatly increased by introducing a heterologous nucleic acid encodingan isoprene synthase polypeptide (e.g., a plant isoprene synthasepolypeptide) into the cells. Isoprene synthase polypeptides convertdimethylallyl diphosphate (DMAPP) into isoprene. As shown in theExamples, a heterologous Pueraria Montana (kudzu) isoprene synthasepolypeptide was expressed in a variety of host cells, such asEscherichia coli, Panteoa citrea, Bacillus subtilis, Yarrowialipolytica, and Trichoderma reesei. All of these cells produced moreisoprene than the corresponding cells without the heterologous isoprenesynthase polypeptide. As illustrated in Tables 1 and 2, large amounts ofisoprene are produced using the methods described herein. For example,B. subtilis cells with a heterologous isoprene synthase nucleic acidproduced approximately 10-fold more isoprene in a 14 liter fermentorthan the corresponding control B. subtilis cells without theheterologous nucleic acid (Table 2). The production of 300 mg ofisoprene per liter of broth (mg/L, wherein the volume of broth includesboth the volume of the cell medium and the volume of the cells) by E.coli and 30 mg/L by B. subtilis in fermentors indicates that significantamounts of isoprene can be generated (Table 2). If desired, isoprene canbe produced on an even larger scale or other conditions described hereincan be used to further increase the amount of isoprene. The vectorslisted in Tables 1 and 2 and the experimental conditions are describedin further detail below and in the Examples section.

TABLE 1 Exemplary yields of isoprene from a shake flask using the cellcultures and methods of the invention. The assay for measuring isopreneproduction is described in Example I, part II. For this assay, a samplewas removed at one or more time points from the shake flask and culturedfor 30 minutes. The amount of isoprene produced in this sample was thenmeasured. The headspace concentration and specific rate of isopreneproduction are listed in Table 1 and described further herein. IsopreneProduction in a Headspace vial* Headspace Specific Rate concentrationμg/L_(broth)/hr/OD Strain μg/L_(gas) (nmol/g_(wcm)/hr) E. coliBL21/pTrcKudzu IS 1.40  53.2 (781.2) E. coli BL21/pCL DXS yidi Kudzu7.61 289.1 IS (4.25 × 10³) E. coli BL21/MCM127 with kudzu 23.0 874.1 ISand entire MVA pathway (12.8 × 10³) E. coli BL21/pET N-HisKudzu IS 1.49 56.6 (831.1) Pantoea citrea/pTrcKudzu IS 0.66  25.1 (368.6) E. coli w/Poplar IS —  5.6 [Miller (2001)]  (82.2) Bacillis licheniformis FallU.S. Pat. No. —  4.2 5,849,970  (61.4) Yarrowia lipolytica with kudzu~0.05 μg/L ~2  isoprene synthase (~30)   Trichoderma reesei with kudzu~0.05 μg/L ~2  isoprene synthase (~30)   E. coli BL21/pTrcKKD_(y)I_(k)ISwith 85.9  3.2 × 10³ kudzu IS and lower MVA pathway  (4.8 × 10⁴)*Normalized to 1 mL of 1 OD₆₀₀, cultured for 1 hour in a sealedheadspace vial with a liquid to headspace volume ratio of 1:19.

TABLE 2 Exemplary yields of isoprene in a fermentor using the cellcultures and methods of the invention. The assay for measuring isopreneproduction is described in Example I, part II. For this assay, a sampleof the off-gas of the fermentor was taken and analyzed for the amount ofisoprene. The peak headspace concentration (which is the highestheadspace concentration during the fermentation), titer (which is thecumulative, total amount of isoprene produced per liter of broth), andpeak specific rate of isoprene production (which is the highest specificrate during the fermentation) are listed in Table 2 and describedfurther herein. Isoprene Production in Fermentors Peak Headspace PeakSpecific rate concentration** Titer μg/L_(broth)/hr/OD Strain(ug/L_(gas)) (mg/L_(broth)) (nmol/g_(wcm)/hr) E. coli BL21/pTrcKudzu 5241.2 37 with Kudzu IS (543.3) E. coli FM5/pTrcKudzu 3 3.5  21.4 IS(308.1) E. coli BL21/triple strain 285 300 240   (DXS, yidi, IS) (3.52 ×10³) E. coli FM5/triple strain 50.8 29 180.8 (DXS, yidi, IS) (2.65 ×10³) E. coli/MCM127 with 3815 3044 992.5 Kudzu IS and entire MVA pathway(1.46 × 10⁴) E. coli BL21/pCLPtrc 2418 1640 1248   UpperPathway gi1.2(1.83 × 10⁴) integrated lower pathway pTrcKudzu E. coli BL21/pCLPtrc3500 3300 1088   UpperPathwayHGS2- (1.60 × 10⁴) pTrcKKDyIkIS Bacillussubtilis wild-type 1.5 2.5  0.8  (11.7) Bacillus pBS Kudzu IS 16.6 ~30 5 (over 100 hrs)  (73.4) Bacillus Marburg 6051 2.04 0.61  24.5 [Wagnerand Fall (1999)] (359.8) Bacillus Marburg 6051 0.7 0.15  6.8 Fall U.S.Pat. No. 5,849,970 (100)   **Normalized to an off-gas flow rate of 1 vvm(1 volume off-gas per 1 L_(broth) per minute).

Additionally, isoprene production by cells that contain a heterologousisoprene synthase nucleic acid can be enhanced by increasing the amountof a 1-deoxy-D-xylulose-5-phosphate synthase (DXS) polypeptide and/or anisopentenyl diphosphate isomerase (IDI) polypeptide expressed by thecells. For example, a DXS nucleic acid and/or an IDI nucleic acid can beintroduced into the cells. The DXS nucleic acid may be a heterologousnucleic acid or a duplicate copy of an endogenous nucleic acid.Similarly, the IDI nucleic acid may be a heterologous nucleic acid or aduplicate copy of an endogenous nucleic acid. In some embodiments, theamount of DXS and/or IDI polypeptide is increased by replacing theendogenous DXS and/or IDI promoters or regulatory regions with otherpromoters and/or regulatory regions that result in greater transcriptionof the DXS and/or IDI nucleic acids. In some embodiments, the cellscontain both a heterologous nucleic acid encoding an isoprene synthasepolypeptide (e.g., a plant isoprene synthase nucleic acid) and aduplicate copy of an endogenous nucleic acid encoding an isoprenesynthase polypeptide.

The encoded DXS and IDI polypeptides are part of the DXP pathway for thebiosynthesis of isoprene (FIG. 19). DXS polypeptides convert pyruvateand D-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate.While not intending to be bound by any particular theory, it is believedthat increasing the amount of DXS polypeptide increases the flow ofcarbon through the DXP pathway, leading to greater isoprene production.IDI polypeptides catalyze the interconversion of isopentenyl diphosphate(IPP) and dimethylallyl diphosphate (DMAPP). While not intending to bebound by any particular theory, it is believed that increasing theamount of IDI polypeptide in cells increases the amount (and conversionrate) of IPP that is converted into DMAPP, which in turn is convertedinto isoprene.

For example, fermentation of E. coli cells with a kudzu isoprenesynthase, S. cerevisia IDI, and E. coli DXS nucleic acids was used toproduce isoprene. The levels of isoprene varied from 50 to 300

g/L over a time period of 15 hours (Example 7, part VII).

In some embodiments, the presence of heterologous or extra endogenousisoprene synthase, IDI, and DXS nucleic acids causes cells to grow morereproducibly or remain viable for longer compared to the correspondingcell with only one or two of these heterologous or extra endogenousnucleic acids. For example, cells containing heterologous isoprenesynthase, IDI, and DXS nucleic acids grew better than cells with onlyheterologous isoprene synthase and DXS nucleic acids or with only aheterologous isoprene synthase nucleic acid. Also, heterologous isoprenesynthase, IDI, and DXS nucleic acids were successfully operably linkedto a strong promoter on a high copy plasmid that was maintained by E.coli cells, suggesting that large amounts of these polypeptides could beexpressed in the cells without causing an excessive amount of toxicityto the cells. While not intending to be bound to a particular theory, itis believed that the presence of heterologous or extra endogenousisoprene synthase and IDI nucleic acids may reduce the amount of one ormore potentially toxic intermediates that would otherwise accumulate ifonly a heterologous or extra endogenous DXS nucleic acid was present inthe cells.

In some embodiments, the production of isoprene by cells by cells thatcontain a heterologous isoprene synthase nucleic acid is augmented byincreasing the amount of a MVA polypeptide expressed by the cells (FIG.19). Exemplary MVA pathways polypeptides include any of the followingpolypeptides: acetyl-CoA acetyltransferase (AA-CoA thiolase)polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase)polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAreductase) polypeptides, mevalonate kinase (MVK) polypeptides,phosphomevalonate kinase (PMK) polypeptides, diphosphomevalontedecarboxylase (MVD) polypeptides, IDI polypeptides, and polypeptides(e.g., fusion polypeptides) having an activity of two or more MVApathway polypeptides. For example, one or more MVA pathway nucleic acidscan be introduced into the cells. In some embodiments, the cells containthe upper MVA pathway, which includes AA-CoA thiolase, HMG-CoA synthase,and HMG-CoA reductase nucleic acids. In some embodiments, the cellscontain the lower MVA pathway, which includes MVK, PMK, MVD, and IDInucleic acids. In some embodiments, the cells contain the entire MVApathway, which includes AA-CoA thiolase, HMG-CoA synthase, HMG-CoAreductase, MVK, PMK, MVD, and IDI nucleic acids. The MVA pathway nucleicacids may be heterologous nucleic acids or duplicate copies ofendogenous nucleic acids. In some embodiments, the amount of one or moreMVA pathway polypeptides is increased by replacing the endogenouspromoters or regulatory regions for the MVA pathway nucleic acids withother promoters and/or regulatory regions that result in greatertranscription of the MVA pathway nucleic acids. In some embodiments, thecells contain both a heterologous nucleic acid encoding an isoprenesynthase polypeptide (e.g., a plant isoprene synthase nucleic acid) anda duplicate copy of an endogenous nucleic acid encoding an isoprenesynthase polypeptide.

For example, E. coli cells containing a nucleic acid encoding a kudzuisoprene synthase polypeptide and nucleic acids encoding Saccharomycescerevisia MVK, PMK, MVD, and IDI polypeptides generated isoprene at arate of 6.67×10⁻⁴ mol/L_(broth)/OD₆₀₀/hr (see Example 8). Additionally,a 14 liter fermentation of E. coli cells with nucleic acids encodingEnterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoAreductase polypeptides produced 22 grams of mevalonic acid (anintermediate of the MVA pathway). A shake flask of these cells produced2-4 grams of mevalonic acid per liter. These results indicate thatheterologous MVA pathways nucleic acids are active in E. coli. E. colicells that contain nucleic acids for both the upper MVA pathway and thelower MVA pathway as well as a kudzu isoprene synthase (strain MCM 127)produced significantly more isoprene (874 ug/L) compared to E. colicells with nucleic acids for only the lower MVA pathway and the kudzuisoprene synthase (strain MCM 131) (see Table 3 and Example 8, partVIII).

In some embodiments, at least a portion of the cells maintain theheterologous isoprene synthase, DXS, IDI, and/or MVA pathway nucleicacid for at least about 5, 10, 20, 50, 75, 100, 200, 300, or more celldivisions in a continuous culture (such as a continuous culture withoutdilution). In some embodiments of any of the aspects of the invention,the nucleic acid comprising the heterologous or duplicate copy of anendogenous isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acidalso comprises a selective marker, such as a kanamycin, ampicillin,carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin,or chloramphenicol antibiotic resistance nucleic acid.

As indicated in Example 7, part VI, the amount of isoprene produced canbe further increased by adding yeast extract to the cell culture medium.In this example, the amount of isoprene produced was linearlyproportional to the amount of yeast extract in the cell medium for theconcentrations tested (FIG. 48C). Additionally, approximately 0.11 gramsof isoprene per liter of broth was produced from a cell medium withyeast extract and glucose (Example 7, part VIII). Both of theseexperiments used E. coli cells with kudzu isoprene synthase, S.cerevisia IDI, and E. coli DXS nucleic acids to produce isoprene.Increasing the amount of yeast extract in the presence of glucoseresulted in more isoprene being produced than increasing the amount ofglucose in the presence of yeast extract. Also, increasing the amount ofyeast extract allowed the cells to produce a high level of isoprene fora longer length of time and improved the health of the cells.

Isoprene production was also demonstrated using three types ofhydrolyzed biomass (bagasse, corn stover, and soft wood pulp) as thecarbon source (FIGS. 46A-C). E. coli cells with kudzu isoprene synthase,S. cerevisia IDI, and E. coli DXS nucleic acids produced as muchisoprene from these hydrolyzed biomass carbon sources as from theequivalent amount of glucose (e.g., 1% glucose, w/v). If desired, anyother biomass carbon source can be used in the compositions and methodsof the invention. Biomass carbon sources are desirable because they arecheaper than many conventional cell mediums, thereby facilitating theeconomical production of isoprene.

Additionally, invert sugar was shown to function as a carbon source forthe generation of isoprene (FIGS. 47C and 96-98). For example, 2.4 g/Lof isoprene was produced from cells expressing MVA pathway polypeptidesand a Kudzu isoprene synthase (Example 8, part XV). Glycerol was as alsoused as a carbon source for the generation of 2.2 mg/L of isoprene fromcells expressing a Kudzu isoprene synthase (Example 8, part XIV).Expressing a DXS nucleic acid, an IDI nucleic acid, and/or one or moreMVA pathway nucleic acids (such as nucleic acids encoding the entire MVApathway) in addition to an isoprene synthase nucleic acid may increasethe production of isoprene from glycerol.

In some embodiments, an oil is included in the cell medium. For example,B. subtilis cells containing a kudzu isoprene synthase nucleic acidproduced isoprene when cultured in a cell medium containing an oil and asource of glucose (Example 4, part III). In some embodiments, more thanone oil (such as 2, 3, 4, 5, or more oils) is included in the cellmedium. While not intending to be bound to any particular theory, it isbelieved that (i) the oil may increase the amount of carbon in the cellsthat is available for conversion to isoprene, (ii) the oil may increasethe amount of acetyl-CoA in the cells, thereby increasing the carbonflow through the MVA pathway, and/or (ii) the oil may provide extranutrients to the cells, which is desirable since a lot of the carbon inthe cells is converted to isoprene rather than other products. In someembodiments, cells that are cultured in a cell medium containing oilnaturally use the MVA pathway to produce isoprene or are geneticallymodified to contain nucleic acids for the entire MVA pathway. In someembodiments, the oil is partially or completely hydrolyzed before beingadded to the cell culture medium to facilitate the use of the oil by thehost cells.

One of the major hurdles to commercial production of small moleculessuch as isoprene in cells (e.g., bacteria) is the decoupling ofproduction of the molecule from growth of the cells. In some embodimentsfor the commercially viable production of isoprene, a signficiant amountof the carbon from the feedstock is converted to isoprene, rather thanto the growth and maintenance of the cells (“carbon efficiency”). Invarious embodiments, the cells convert greater than or about 0.0015,0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5,4.0, 5.0, 6.0, 7.0, or 8.0% of the carbon in the cell culture mediuminto isoprene. In particular embodiments, a significant portion of thecarbon from the feedstock that is converted to downstream products isconverted to isoprene. As described further in Example 11, E. coli cellsexpressing MVA pathway and kudzu isoprene synthase nucleic acidsexhibited decoupling of the production of isoprene or the intermediatemevalonic acid from growth, resulting in high carbon efficiency. Inparticular, mevalonic acid was formed from cells expressing the upperMVA pathway from Enterococcus faecalis. Isoprene was formed from cellsexpressing the upper MVA pathway from Enterococcus faecalis, the lowerMVA pathway from Saccharomyces cerevisiae, and the isoprene synthasefrom Pueraria montana (Kudzu). This decoupling of isoprene or mevalonicacid production from growth was demonstrated in four different strainsof E. coli: BL21(LDE3), BL21(LDE3) Tuner, FMS, and MG1655. The first twoE. coli strains are B strains, and the latter two are K12 strains.Decoupling of production from growth was also demonstrated in a variantof MG1655 with ack and pta genes deleted. This variant also demonstratedless production of acetate.

Exemplary Polypeptides and Nucleic Acids

Various isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides andnucleic acids can be used in the compositions and methods of theinvention.

As used herein, “polypeptides” includes polypeptides, proteins,peptides, fragments of polypeptides, and fusion polypeptides. In someembodiments, the fusion polypeptide includes part or all of a firstpolypeptide (e.g., an isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide or catalytically active fragment thereof) and may optionallyinclude part or all of a second polypeptide (e.g., a peptide thatfacilitates purification or detection of the fusion polypeptide, such asa His-tag). In some embodiments, the fusion polypeptide has an activityof two or more MVA pathway polypeptides (such as AA-CoA thiolase andHMG-CoA reductase polypeptides). In some embodiments, the polypeptide isa naturally-occurring polypeptide (such as the polypeptide encoded by anEnterococcus faecalis mvaE nucleic acid) that has an activity of two ormore MVA pathway polypeptides.

In various embodiments, a polypeptide has at least or about 50, 100,150, 175, 200, 250, 300, 350, 400, or more amino acids. In someembodiments, the polypeptide fragment contains at least or about 25, 50,75, 100, 150, 200, 300, or more contiguous amino acids from afull-length polypeptide and has at least or about 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of an activity of acorresponding full-length polypeptide. In particular embodiments, thepolypeptide includes a segment of or the entire amino acid sequence ofany naturally-occurring isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide. In some embodiments, the polypeptide has one or moremutations compared to the sequence of a wild-type (i.e., a sequenceoccurring in nature) isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide.

In some embodiments, the polypeptide is an isolated polypeptide. As usedherein, an “isolated polypeptide” is not part of a library ofpolypeptides, such as a library of 2, 5, 10, 20, 50 or more differentpolypeptides and is separated from at least one component with which itoccurs in nature. An isolated polypeptide can be obtained, for example,by expression of a recombinant nucleic acid encoding the polypeptide.

In some embodiments, the polypeptide is a heterologous polypeptide. By“heterologous polypeptide” is meant a polypeptide whose amino acidsequence is not identical to that of another polypeptide naturallyexpressed in the same host cell. In particular, a heterologouspolypeptide is not identical to a wild-type nucleic acid that is foundin the same host cell in nature.

As used herein, a “nucleic acid” refers to two or moredeoxyribonucleotides and/or ribonucleotides in either single ordouble-stranded form. In some embodiments, the nucleic acid is arecombinant nucleic acid. By “recombinant nucleic acid” means a nucleicacid of interest that is free of one or more nucleic acids (e.g., genes)which, in the genome occurring in nature of the organism from which thenucleic acid of interest is derived, flank the nucleic acid of interest.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector, into an autonomously replicating plasmid orvirus, or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA, a genomic DNA fragment, ora cDNA fragment produced by PCR or restriction endonuclease digestion)independent of other sequences. In various embodiments, a nucleic acidis a recombinant nucleic acid. In some embodiments, an isoprenesynthase, DXS, IDI, or MVA pathway nucleic acid is operably linked toanother nucleic acid encoding all or a portion of another polypeptidesuch that the recombinant nucleic acid encodes a fusion polypeptide thatincludes an isoprene synthase, DXS, IDI, or MVA pathway polypeptide andall or part of another polypeptide (e.g., a peptide that facilitatespurification or detection of the fusion polypeptide, such as a His-tag).In some embodiments, part or all of a recombinant nucleic acid ischemically synthesized.

In some embodiments, the nucleic acid is a heterologous nucleic acid. By“heterologous nucleic acid” is meant a nucleic acid whose nucleic acidsequence is not identical to that of another nucleic acid naturallyfound in the same host cell.

In particular embodiments, the nucleic acid includes a segment of or theentire nucleic acid sequence of any naturally-occurring isoprenesynthase, DXS, IDI, or MVA pathway nucleic acid. In some embodiments,the nucleic acid includes at least or about 50, 100, 150, 200, 300, 400,500, 600, 700, 800, or more contiguous nucleotides from anaturally-occurring isoprene synthase nucleic acid DXS, IDI, or MVApathway nucleic acid. In some embodiments, the nucleic acid has one ormore mutations compared to the sequence of a wild-type (i.e., a sequenceoccurring in nature) isoprene synthase, DXS, IDI, or MVA pathway nucleicacid. In some embodiments, the nucleic acid has one or more mutations(e.g., a silent mutation) that increase the transcription or translationof isoprene synthase, DXS, IDI, or MVA pathway nucleic acid. In someembodiments, the nucleic acid is a degenerate variant of any nucleicacid encoding an isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a nucleic acid for improved expression in a host cell, itis desirable in some embodiments to design the nucleic acid such thatits frequency of codon usage approaches the frequency of preferred codonusage of the host cell.

The accession numbers of exemplary isoprene synthase, DXS, IDI, and/orMVA pathway polypeptides and nucleic acids are listed in Appendix 1 (theaccession numbers of Appendix 1 and their corresponding sequences areherein incorporated by reference in their entireties, particularly withrespect to the amino acid and nucleic acid sequences of isoprenesynthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids).The Kegg database also contains the amino acid and nucleic acidsequences of numerous exemplary isoprene synthase, DXS, IDI, and/or MVApathway polypeptides and nucleic acids (see, for example, the world-wideweb at “genome.jp/kegg/pathway/map/map00100.html” and the sequencestherein, which are each hereby incorporated by reference in theirentireties, particularly with respect to the amino acid and nucleic acidsequences of isoprene synthase, DXS, IDI, and/or MVA pathwaypolypeptides and nucleic acids). In some embodiments, one or more of theisoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and/ornucleic acids have a sequence identical to a sequence publicly availableon Dec. 12, 2007, such as any of the sequences that correspond to any ofthe accession numbers in Appendix 1 or any of the sequences present inthe Kegg database. Additional exemplary isoprene synthase, DXS, IDI,and/or MVA pathway polypeptides and nucleic acids are described furtherbelow.

Exemplary Isoprene Synthase Polypeptides and Nucleic Acids

As noted above, isoprene synthase polypeptides convert dimethylallyldiphosphate (DMAPP) into isoprene. Exemplary isoprene synthasepolypeptides include polypeptides, fragments of polypeptides, peptides,and fusions polypeptides that have at least one activity of an isoprenesynthase polypeptide. Standard methods can be used to determine whethera polypeptide has isoprene synthase polypeptide activity by measuringthe ability of the polypeptide to convert DMAPP into isoprene in vitro,in a cell extract, or in vivo. In an exemplary assay, cell extracts areprepared by growing a strain (e.g., the E. coli/pTrcKudzu straindescribed herein) in the shake flask method as described in Example 1.After induction is complete, approximately 10 mL of cells are pelletedby centrifugation at 7000×g for 10 minutes and resuspended in 5 ml ofPEB without glycerol. The cells are lysed using a French Pressure cellusing standard procedures. Alternatively the cells are treated withlysozyme (Ready-Lyse lysozyme solution; EpiCentre) after a freeze/thawat −80 C.

Isoprene synthase polypeptide activity in the cell extract can bemeasured, for example, as described in Silver et al., J. Biol. Chem.270:13010-13016, 1995 and references therein, which are each herebyincorporated by reference in their entireties, particularly with respectto assays for isoprene synthase polypeptide activity. DMAPP (Sigma) isevaporated to dryness under a stream of nitrogen and rehydrated to aconcentration of 100 mM in 100 mM potassium phosphate buffer pH 8.2 andstored at −20° C. To perform the assay, a solution of 5 μL of 1M MgCl₂,1 mM (250 μg/ml) DMAPP, 65 μL of Plant Extract Buffer (PEB) (50 mMTris-HCl, pH 8.0, 20 mM MgCl₂, 5% glycerol, and 2 mM DTT) is added to 25μL of cell extract in a 20 ml Headspace vial with a metal screw cap andteflon coated silicon septum (Agilent Technologies) and cultured at 37°C. for 15 minutes with shaking. The reaction is quenched by adding 200μL of 250 mM EDTA and quantified by GC/MS as described in Example 1,part II.

Exemplary isoprene synthase nucleic acids include nucleic acids thatencode a polypeptide, fragment of a polypeptide, peptide, or fusionpolypeptide that has at least one activity of an isoprene synthasepolypeptide. Exemplary isoprene synthase polypeptides and nucleic acidsinclude naturally-occurring polypeptides and nucleic acids from any ofthe source organisms described herein as well as mutant polypeptides andnucleic acids derived from any of the source organisms described herein.

In some embodiments, the isoprene synthase polypeptide or nucleic acidis from the family Fabaceae, such as the Faboideae subfamily. In someembodiments, the isoprene synthase polypeptide or nucleic acid is anaturally-occurring polypeptide or nucleic acid from Pueraria montana(kudzu) (Sharkey et al., Plant Physiology 137: 700-712, 2005), Puerarialobata, poplar (such as Populus alba×tremula CAC35696) Miller et al.,Planta 213: 483-487, 2001) aspen (such as Populus tremuloides) Silver etal., JBC 270(22): 13010-1316, 1995), or English Oak (Quercus robur)(Zimmer et al., WO 98/02550), which are each hereby incorporated byreference in their entireties, particularly with respect to isoprenesynthase nucleic acids and the expression of isoprene synthasepolypeptides. Suitable isoprene synthases include, but are not limitedto, those identified by Genbank Accession Nos. AY341431, AY316691,AY279379, AJ457070, and AY182241, which are each hereby incorporated byreference in their entireties, particularly with respect to sequences ofisoprene synthase nucleic acids and polypeptides. In some embodiments,the isoprene synthase polypeptide or nucleic acid is not anaturally-occurring polypeptide or nucleic acid from Quercus robur(i.e., the isoprene synthase polypeptide or nucleic acid is an isoprenesynthase polypeptide or nucleic acid other than a naturally-occurringpolypeptide or nucleic acid from Quercus robur). In some embodiments,the isoprene synthase nucleic acid or polypeptide is not anaturally-occurring polypeptide or nucleic acid from poplar (such asPopulus alba×tremula CAC35696).

Exemplary DXS Polypeptides and Nucleic Acids

As noted above, 1-deoxy-D-xylulose-5-phosphate synthase (DXS)polypeptides convert pyruvate and D-glyceraldehyde-3-phosphate into1-deoxy-D-xylulose-5-phosphate. Exemplary DXS polypeptides includepolypeptides, fragments of polypeptides, peptides, and fusionspolypeptides that have at least one activity of a DXS polypeptide.Standard methods (such as those described herein) can be used todetermine whether a polypeptide has DXS polypeptide activity bymeasuring the ability of the polypeptide to convert pyruvate andD-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate invitro, in a cell extract, or in vivo. Exemplary DXS nucleic acidsinclude nucleic acids that encode a polypeptide, fragment of apolypeptide, peptide, or fusion polypeptide that has at least oneactivity of a DXS polypeptide. Exemplary DXS polypeptides and nucleicacids include naturally-occurring polypeptides and nucleic acids fromany of the source organisms described herein as well as mutantpolypeptides and nucleic acids derived from any of the source organismsdescribed herein.

Exemplary IDI Polypeptides and Nucleic Acids

Isopentenyl diphosphate isomerase polypeptides (isopentenyl-diphosphatedelta-isomerase or IDI) catalyses the interconversion of isopentenyldiphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (e.g.,converting IPP into DMAPP and/or converting DMAPP into IPP). ExemplaryIDI polypeptides include polypeptides, fragments of polypeptides,peptides, and fusions polypeptides that have at least one activity of anIDI polypeptide. Standard methods (such as those described herein) canbe used to determine whether a polypeptide has IDI polypeptide activityby measuring the ability of the polypeptide to interconvert IPP andDMAPP in vitro, in a cell extract, or in vivo. Exemplary IDI nucleicacids include nucleic acids that encode a polypeptide, fragment of apolypeptide, peptide, or fusion polypeptide that has at least oneactivity of an IDI polypeptide. Exemplary IDI polypeptides and nucleicacids include naturally-occurring polypeptides and nucleic acids fromany of the source organisms described herein as well as mutantpolypeptides and nucleic acids derived from any of the source organismsdescribed herein.

Exemplary MVA Pathway Polypeptides and Nucleic Acids

Exemplary MVA pathway polypeptides include acetyl-CoA acetyltransferase(AA-CoA thiolase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase(HMG-CoA synthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoAreductase (HMG-CoA reductase) polypeptides, mevalonate kinase (MVK)polypeptides, phosphomevalonate kinase (PMK) polypeptides,diphosphomevalonte decarboxylase (MVD) polypeptides, IDI polypeptides,and polypeptides (e.g., fusion polypeptides) having an activity of twoor more MVA pathway polypeptides. In particular, MVA pathwaypolypeptides include polypeptides, fragments of polypeptides, peptides,and fusions polypeptides that have at least one activity of an MVApathway polypeptide. Exemplary MVA pathway nucleic acids include nucleicacids that encode a polypeptide, fragment of a polypeptide, peptide, orfusion polypeptide that has at least one activity of an MVA pathwaypolypeptide. Exemplary MVA pathway polypeptides and nucleic acidsinclude naturally-occurring polypeptides and nucleic acids from any ofthe source organisms described herein as well as mutant polypeptides andnucleic acids derived from any of the source organisms described herein.

In particular, acetyl-CoA acetyltransferase polypeptides (AA-CoAthiolase or AACT) convert two molecules of acetyl-CoA intoacetoacetyl-CoA. Standard methods (such as those described herein) canbe used to determine whether a polypeptide has AA-CoA thiolasepolypeptide activity by measuring the ability of the polypeptide toconvert two molecules of acetyl-CoA into acetoacetyl-CoA in vitro, in acell extract, or in vivo.

3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase or HMGS)polypeptides convert acetoacetyl-CoA into3-hydroxy-3-methylglutaryl-CoA. Standard methods (such as thosedescribed herein) can be used to determine whether a polypeptide hasHMG-CoA synthase polypeptide activity by measuring the ability of thepolypeptide to convert acetoacetyl-CoA into3-hydroxy-3-methylglutaryl-CoA in vitro, in a cell extract, or in vivo.

3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase or HMGR)polypeptides convert 3-hydroxy-3-methylglutaryl-CoA into mevalonate.Standard methods (such as those described herein) can be used todetermine whether a polypeptide has HMG-CoA reductase polypeptideactivity by measuring the ability of the polypeptide to convert3-hydroxy-3-methylglutaryl-CoA into mevalonate in vitro, in a cellextract, or in vivo.

Mevalonate kinase (MVK) polypeptides phosphorylates mevalonate to formmevalonate-5-phosphate. Standard methods (such as those describedherein) can be used to determine whether a polypeptide has MVKpolypeptide activity by measuring the ability of the polypeptide toconvert mevalonate into mevalonate-5-phosphate in vitro, in a cellextract, or in vivo.

Phosphomevalonate kinase (PMK) polypeptides phosphorylatesmevalonate-5-phosphate to form mevalonate-5-diphosphate. Standardmethods (such as those described herein) can be used to determinewhether a polypeptide has PMK polypeptide activity by measuring theability of the polypeptide to convert mevalonate-5-phosphate intomevalonate-5-diphosphate in vitro, in a cell extract, or in vivo.

Diphosphomevalonte decarboxylase (MVD or DPMDC) polypeptides convertmevalonate-5-diphosphate into isopentenyl diphosphate polypeptides(IPP). Standard methods (such as those described herein) can be used todetermine whether a polypeptide has MVD polypeptide activity bymeasuring the ability of the polypeptide to convertmevalonate-5-diphosphate into IPP in vitro, in a cell extract, or invivo.

Exemplary IDI polypeptides and nucleic acids are described above.

Exemplary Methods for Isolating Nucleic Acids

Isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids can beisolated using standard methods. Methods of obtaining desired nucleicacids from a source organism of interest (such as a bacterial genome)are common and well known in the art of molecular biology (see, forexample, WO 2004/033646 and references cited therein, which are eachhereby incorporated by reference in their entireties, particularly withrespect to the isolation of nucleic acids of interest). For example, ifthe sequence of the nucleic acid is known (such as any of the knownnucleic acids described herein), suitable genomic libraries may becreated by restriction endonuclease digestion and may be screened withprobes complementary to the desired nucleic acid sequence. Once thesequence is isolated, the DNA may be amplified using standard primerdirected amplification methods such as polymerase chain reaction (PCR)(U.S. Pat. No. 4,683,202, which is incorporated by reference in itsentirety, particularly with respect to PCR methods) to obtain amounts ofDNA suitable for transformation using appropriate vectors.

Alternatively, isoprene synthase, DXS, IDI, and/or MVA pathway nucleicacids (such as any isoprene synthase, DXS, IDI, and/or MVA pathwaynucleic acids with a known nucleic acid sequence) can be chemicallysynthesized using standard methods.

Additional isoprene synthase, DXS, IDI, or MVA pathway polypeptides andnucleic acids which may be suitable for use in the compositions andmethods described herein can be identified using standard methods. Forexample, cosmid libraries of the chromosomal DNA of organisms known toproduce isoprene naturally can be constructed in organisms such as E.coli, and then screened for isoprene production. In particular, cosmidlibraries may be created where large segments of genomic DNA (35-45 kb)are packaged into vectors and used to transform appropriate hosts.Cosmid vectors are unique in being able to accommodate large quantitiesof DNA. Generally cosmid vectors have at least one copy of the cos DNAsequence which is needed for packaging and subsequent circularization ofthe heterologous DNA. In addition to the cos sequence, these vectorsalso contain an origin of replication such as ColEI and drug resistancemarkers such as a nucleic acid resistant to ampicillin or neomycin.Methods of using cosmid vectors for the transformation of suitablebacterial hosts are well described in Sambrook et al., MolecularCloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989,which is hereby incorporated by reference in its entirety, particularlywith respect to transformation methods.

Typically to clone cosmids, heterologous DNA is isolated using theappropriate restriction endonucleases and ligated adjacent to the cosregion of the cosmid vector using the appropriate ligases. Cosmidvectors containing the linearized heterologous DNA are then reacted witha DNA packaging vehicle such as bacteriophage. During the packagingprocess, the cos sites are cleaved and the heterologous DNA is packagedinto the head portion of the bacterial viral particle. These particlesare then used to transfect suitable host cells such as E. coli. Onceinjected into the cell, the heterologous DNA circularizes under theinfluence of the cos sticky ends. In this manner, large segments ofheterologous DNA can be introduced and expressed in host cells.

Additional methods for obtaining isoprene synthase, DXS, IDI, and/or MVApathway nucleic acids include screening a metagenomic library by assay(such as the headspace assay described herein) or by PCR using primersdirected against nucleotides encoding for a length of conserved aminoacids (for example, at least 3 conserved amino acids). Conserved aminoacids can be identified by aligning amino acid sequences of knownisoprene synthase, DXS, IDI, and/or MVA pathway polypeptides. Conservedamino acids for isoprene synthase polypeptides can be identified basedon aligned sequences of known isoprene synthase polypeptides. Anorganism found to produce isoprene naturally can be subjected tostandard protein purification methods (which are well known in the art)and the resulting purified polypeptide can be sequenced using standardmethods. Other methods are found in the literature (see, for example,Julsing et al., Applied. Microbiol. Biotechnol. 75: 1377-84, 2007;Withers et al., Appl Environ Microbiol. 73(19):6277-83, 2007, which areeach hereby incorporated by reference in their entireties, particularlywith respect to identification of nucleic acids involved in thesynthesis of isoprene).

Additionally, standard sequence alignment and/or structure predictionprograms can be used to identify additional DXS, IDI, or MVA pathwaypolypeptides and nucleic acids based on the similarity of their primaryand/or predicted polypeptide secondary structure with that of known DXS,IDI, or MVA pathway polypeptides and nucleic acids. Standard databasessuch as the swissprot-trembl database (world-wide web at “expasy.org”,Swiss Institute of Bioinformatics Swiss-Prot group CMU-1 rue MichelServet CH-1211 Geneva 4, Switzerland) can also be used to identifyisoprene synthase, DXS, IDI, or MVA pathway polypeptides and nucleicacids. The secondary and/or tertiary structure of an isoprene synthase,DXS, IDI, or MVA pathway polypeptide can be predicted using the defaultsettings of standard structure prediction programs, such asPredictProtein (630 West, 168 Street, BB217, New York, N.Y. 10032, USA).Alternatively, the actual secondary and/or tertiary structure of anisoprene synthase, DXS, IDI, or MVA pathway polypeptide can bedetermined using standard methods. Additional isoprene synthase, DXS,IDI, or MVA pathway nucleic acids can also be identified byhybridization to probes generated from known isoprene synthase, DXS,IDI, or MVA pathway nucleic acids.

Exemplary Promoters and Vectors

Any of the isoprene synthase, DXS, IDI, or MVA pathway nucleic aciddescribed herein can be included in one or more vectors. Accordingly,the invention also features vectors with one more nucleic acids encodingany of the isoprene synthase, DXS, IDI, or MVA pathway polypeptides thatare described herein. As used herein, a “vector” means a construct thatis capable of delivering, and desirably expressing one or more nucleicacids of interest in a host cell. Examples of vectors include, but arenot limited to, plasmids, viral vectors, DNA or RNA expression vectors,cosmids, and phage vectors. In some embodiments, the vector contains anucleic acid under the control of an expression control sequence.

As used herein, an “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid of interest. Anexpression control sequence can be a promoter, such as a constitutive oran inducible promoter, or an enhancer. An “inducible promoter” is apromoter that is active under environmental or developmental regulation.The expression control sequence is operably linked to the nucleic acidsegment to be transcribed.

In some embodiments, the vector contains a selective marker. The term“selective marker” refers to a nucleic acid capable of expression in ahost cell that allows for ease of selection of those host cellscontaining an introduced nucleic acid or vector. Examples of selectablemarkers include, but are not limited to, antibiotic resistance nucleicacids (e.g., kanamycin, ampicillin, carbenicillin, gentamicin,hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol) and/ornucleic acids that confer a metabolic advantage, such as a nutritionaladvantage on the host cell. Exemplary nutritional selective markersinclude those markers known in the art as amdS, argB, and pyr4. Markersuseful in vector systems for transformation of Trichoderma are known inthe art (see, e.g., Finkelstein, Chapter 6 in Biotechnology ofFilamentous Fungi, Finkelstein et al., Eds. Butterworth-Heinemann,Boston, Mass., Chap. 6., 1992; and Kinghorn et al., Applied MolecularGenetics of Filamentous Fungi, Blackie Academic and Professional,Chapman and Hall, London, 1992, which are each hereby incorporated byreference in their entireties, particularly with respect to selectivemarkers). In some embodiments, the selective marker is the amdS nucleicacid, which encodes the enzyme acetamidase, allowing transformed cellsto grow on acetamide as a nitrogen source. The use of an A. nidulansamdS nucleic acid as a selective marker is described in Kelley et al.,EMBO J. 4:475-479, 1985 and Penttila et al., Gene 61:155-164, 1987(which are each hereby incorporated by reference in their entireties,particularly with respect to selective markers). In some embodiments, anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid integrates intoa chromosome of the cells without a selective marker.

Suitable vectors are those which are compatible with the host cellemployed. Suitable vectors can be derived, for example, from abacterium, a virus (such as bacteriophage T7 or a M-13 derived phage), acosmid, a yeast, or a plant. Protocols for obtaining and using suchvectors are known to those in the art (see, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor, 1989, which is hereby incorporated by reference in its entirety,particularly with respect to the use of vectors).

Promoters are well known in the art. Any promoter that functions in thehost cell can be used for expression of an isoprene synthase, DXS, IDI,or MVA pathway nucleic acid in the host cell. Initiation control regionsor promoters, which are useful to drive expression of isoprene synthase,DXS, IDI, or MVA pathway nucleic acids in various host cells arenumerous and familiar to those skilled in the art (see, for example, WO2004/033646 and references cited therein, which are each herebyincorporated by reference in their entireties, particularly with respectto vectors for the expression of nucleic acids of interest). Virtuallyany promoter capable of driving these nucleic acids is suitable for thepresent invention including, but not limited to, CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHOS, GAPDH, ADCI, TRP1, URA3, LEU2, ENO, and TPI(useful for expression in Saccharomyces); AOX1 (useful for expression inPichia); and lac, trp,

P_(L),

P_(R), T7, tac, and trc (useful for expression in E. coli).

In some embodiments, a glucose isomerase promoter is used (see, forexample, U.S. Pat. No. 7,132,527 and references cited therein, which areeach hereby incorporated by reference in their entireties, particularlywith respect promoters and plasmid systems for expressing polypeptidesof interest). Reported glucose isomerase promoter mutants can be used tovary the level of expression of the polypeptide encoded by a nucleicacid operably linked to the glucose isomerase promoter (U.S. Pat. No.7,132,527). In various embodiments, the glucose isomerase promoter iscontained in a low, medium, or high copy plasmid (U.S. Pat. No.7,132,527).

In various embodiments, an isoprene synthase, DXS, IDI, and/or MVApathway nucleic acid is contained in a low copy plasmid (e.g., a plasmidthat is maintained at about 1 to about 4 copies per cell), medium copyplasmid (e.g., a plasmid that is maintained at about 10 to about 15copies per cell), or high copy plasmid (e.g., a plasmid that ismaintained at about 50 or more copies per cell). In some embodiments,the heterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a T7 promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a T7 promoteris contained in a medium or high copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a Trc promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a Trc promoteris contained in a medium or high copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a Lac promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a Lac promoteris contained in a low copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to an endogenous promoter, suchas an endogenous Escherichia, Panteoa, Bacillus, Yarrowia, Streptomyces,or Trichoderma promoter or an endogenous alkaline serine protease,isoprene synthase, DXS, IDI, or MVA pathway promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to an endogenouspromoter is contained in a high copy plasmid. In some embodiments, thevector is a replicating plasmid that does not integrate into achromosome in the cells. In some embodiments, part or all of the vectorintegrates into a chromosome in the cells.

In some embodiments, the vector is any vector which when introduced intoa fungal host cell is integrated into the host cell genome and isreplicated. Reference is made to the Fungal Genetics Stock CenterCatalogue of Strains (FGSC, the world-wide web at “fgsc.net” and thereferences cited therein, which are each hereby incorporated byreference in their entireties, particularly with respect to vectors) fora list of vectors. Additional examples of suitable expression and/orintegration vectors are provided in Sambrook et al., Molecular Cloning:A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989, CurrentProtocols in Molecular Biology (F. M. Ausubel et al. (eds) 1987,Supplement 30, section 7.7.18); van den Hondel et al. in Bennett andLasure (Eds.) More Gene Manipulations in Fungi, Academic Press pp.396-428, 1991; and U.S. Pat. No. 5,874,276, which are each herebyincorporated by reference in their entireties, particularly with respectto vectors. Particularly useful vectors include pFB6, pBR322, PUC18,pUC100, and pENTR/D.

In some embodiments, an isoprene synthase, DXS, IDI, or MVA pathwaynucleic acid is operably linked to a suitable promoter that showstranscriptional activity in a fungal host cell. The promoter may bederived from one or more nucleic acids encoding a polypeptide that iseither endogenous or heterologous to the host cell. In some embodiments,the promoter is useful in a Trichoderma host. Suitable non-limitingexamples of promoters include cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2,xynl, and amy. In some embodiments, the promoter is one that is nativeto the host cell. For example, in some embodiments when T. reesei is thehost, the promoter is a native T. reesei promoter. In some embodiments,the promoter is T. reesei cbh1, which is an inducible promoter and hasbeen deposited in GenBank under Accession No. D86235, which isincorporated by reference in its entirety, particularly with respect topromoters. In some embodiments, the promoter is one that is heterologousto the fungal host cell. Other examples of useful promoters includepromoters from the genes of A. awamori and A. niger glucoamylase (glaA)(Nunberg et al., Mol. Cell. Biol. 4:2306-2315, 1984 and Boel et al.,EMBO J. 3:1581-1585, 1984, which are each hereby incorporated byreference in their entireties, particularly with respect to promoters);Aspergillus niger alpha amylases, Aspergillus oryzae TAKA amylase, T.reesei xlnl, and the T. reesei cellobiohydrolase 1 (EP 137280, which isincorporated by reference in its entirety, particularly with respect topromoters).

In some embodiments, the expression vector also includes a terminationsequence. Termination control regions may also be derived from variousgenes native to the host cell. In some embodiments, the terminationsequence and the promoter sequence are derived from the same source. Inanother embodiment, the termination sequence is endogenous to the hostcell. A particularly suitable terminator sequence is cbh1 derived from aTrichoderma strain (such as T. reesei). Other useful fungal terminatorsinclude the terminator from an A. niger or A. awamori glucoamylasenucleic acid (Nunberg et al., Mol. Cell. Biol. 4:2306-2315, 1984 andBoel et al., EMBO J. 3:1581-1585, 1984; which are each herebyincorporated by reference in their entireties, particularly with respectto fungal terminators). Optionally, a termination site may be included.For effective expression of the polypeptides, DNA encoding thepolypeptide are linked operably through initiation codons to selectedexpression control regions such that expression results in the formationof the appropriate messenger RNA.

In some embodiments, the promoter, coding, region, and terminator alloriginate from the isoprene synthase, DXS, IDI, or MVA pathway nucleicacid to be expressed. In some embodiments, the coding region for anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid is insertedinto a general-purpose expression vector such that it is under thetranscriptional control of the expression construct promoter andterminator sequences. In some embodiments, genes or part thereof areinserted downstream of the strong cbh1 promoter.

An isoprene synthase, DXS, IDI, or MVA pathway nucleic acid can beincorporated into a vector, such as an expression vector, using standardtechniques (Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, 1982, which is hereby incorporated by reference inits entirety, particularly with respect to the screening of appropriateDNA sequences and the construction of vectors). Methods used to ligatethe DNA construct comprising a nucleic acid of interest (such as anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid), a promoter, aterminator, and other sequences and to insert them into a suitablevector are well known in the art. For example, restriction enzymes canbe used to cleave the isoprene synthase, DXS, IDI, or MVA pathwaynucleic acid and the vector. Then, the compatible ends of the cleavedisoprene synthase, DXS, IDI, or MVA pathway nucleic acid and the cleavedvector can be ligated. Linking is generally accomplished by ligation atconvenient restriction sites. If such sites do not exist, the syntheticoligonucleotide linkers are used in accordance with conventionalpractice (see, Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) ed., Cold Spring Harbor, 1989, and Bennett and Lasure, More GeneManipulations in Fungi, Academic Press, San Diego, pp 70-76, 1991, whichare each hereby incorporated by reference in their entireties,particularly with respect to oligonucleotide linkers). Additionally,vectors can be constructed using known recombination techniques (e.g.,Invitrogen Life Technologies, Gateway Technology).

In some embodiments, it may be desirable to over-express isoprenesynthase, DXS, IDI, or MVA pathway nucleic acids at levels far higherthan currently found in naturally-occurring cells. This result may beaccomplished by the selective cloning of the nucleic acids encodingthose polypeptides into multicopy plasmids or placing those nucleicacids under a strong inducible or constitutive promoter. Methods forover-expressing desired polypeptides are common and well known in theart of molecular biology and examples may be found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor,1989, which is hereby incorporated by reference in its entirety,particularly with respect to cloning techniques.

The following resources include descriptions of additional generalmethodology useful in accordance with the invention: Kreigler, GeneTransfer and Expression; A Laboratory Manual, 1990 and Ausubel et al.,Eds. Current Protocols in Molecular Biology, 1994, which are each herebyincorporated by reference in their entireties, particularly with respectto molecular biology and cloning techniques.

Exemplary Source Organisms

Isoprene synthase, DXS, IDI, or MVA pathway nucleic acids (and theirencoded polypeptides) can be obtained from any organism that naturallycontains isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids.As noted above, isoprene is formed naturally by a variety of organisms,such as bacteria, yeast, plants, and animals. Organisms contain the MVApathway, DXP pathway, or both the MVA and DXP pathways for producingisoprene (FIG. 19). Thus, DXS nucleic acids can be obtained, e.g., fromany organism that contains the DXP pathway or contains both the MVA andDXP pathways. IDI and isoprene synthase nucleic acids can be obtained,e.g., from any organism that contains the MVA pathway, DXP pathway, orboth the MVA and DXP pathways. MVA pathway nucleic acids can beobtained, e.g., from any organism that contains the MVA pathway orcontains both the MVA and DXP pathways.

In some embodiments, the nucleic acid sequence of the isoprene synthase,DXS, IDI, or MVA pathway nucleic is identical to the sequence of anucleic acid that is produced by any of the following organisms innature. In some embodiments, the amino acid sequence of the isoprenesynthase, DXS, IDI, or MVA pathway polypeptide is identical to thesequence of a polypeptide that is produced by any of the followingorganisms in nature. In some embodiments, the isoprene synthase, DXS,IDI, or MVA pathway nucleic acid or polypeptide is a mutant nucleic acidor polypeptide derived from any of the organisms described herein. Asused herein, “derived from” refers to the source of the nucleic acid orpolypeptide into which one or more mutations is introduced. For example,a polypeptide that is “derived from a plant polypeptide” refers topolypeptide of interest that results from introducing one or moremutations into the sequence of a wild-type (i.e., a sequence occurringin nature) plant polypeptide.

In some embodiments, the source organism is a fungus, examples of whichare species of Aspergillus such as A. oryzae and A. niger, species ofSaccharomyces such as S. cerevisiae, species of Schizosaccharomyces suchas S. pombe, and species of Trichoderma such as T. reesei. In someembodiments, the source organism is a filamentous fungal cell. The term“filamentous fungi” refers to all filamentous forms of the subdivisionEumycotina (see, Alexopoulos, C. J. (1962), Introductory Mycology,Wiley, New York). These fungi are characterized by a vegetative myceliumwith a cell wall composed of chitin, cellulose, and other complexpolysaccharides. The filamentous fungi are morphologically,physiologically, and genetically distinct from yeasts. Vegetative growthby filamentous fungi is by hyphal elongation and carbon catabolism isobligatory aerobic. The filamentous fungal parent cell may be a cell ofa species of, but not limited to, Trichoderma, (e.g., Trichodermareesei, the asexual morph of Hypocrea jecorina, previously classified asT. longibrachiatum, Trichoderma viride, Trichoderma koningii,Trichoderma harzianum) (Sheir-Neirs et al., Appl. Microbiol. Biotechnol20: 46-53, 1984; ATCC No. 56765 and ATCC No. 26921); Penicillium sp.,Humicola sp. (e.g., H. insolens, H. lanuginose, or H. grisea);Chrysosporium sp. (e.g., C. lucknowense), Gliocladium sp., Aspergillussp. (e.g., A. oryzae, A. niger, A sojae, A. japonicus, A. nidulans, orA. awamori) (Ward et al., Appl. Microbiol. Biotechnol. 39: 7380743, 1993and Goedegebuur et al., Genet. 41: 89-98, 2002), Fusarium sp., (e.g., F.roseum, F. graminum F. cerealis, F. oxysporuim, or F. venenatum),Neurospora sp., (e.g., N. crassa), Hypocrea sp., Mucor sp., (e.g., M.miehei), Rhizopus sp. and Emericella sp. (see also, Innis et al., Sci.228: 21-26, 1985). The term “Trichoderma” or “Trichoderma sp.” or“Trichoderma spp.” refer to any fungal genus previously or currentlyclassified as Trichoderma.

In some embodiments, the fungus is A. nidulans, A. awamori, A. oryzae,A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F.oxysporum, or F. solani. Aspergillus strains are disclosed in Ward etal., Appl. Microbiol. Biotechnol. 39:738-743, 1993 and Goedegebuur etal., Curr Gene 41:89-98, 2002, which are each hereby incorporated byreference in their entireties, particularly with respect to fungi. Inparticular embodiments, the fungus is a strain of Trichoderma, such as astrain of T. reesei. Strains of T. reesei are known and non-limitingexamples include ATCC No. 13631, ATCC No. 26921, ATCC No. 56764, ATCCNo. 56765, ATCC No. 56767, and NRRL 15709, which are each herebyincorporated by reference in their entireties, particularly with respectto strains of T. reesei. In some embodiments, the host strain is aderivative of RL-P37. RL-P37 is disclosed in Sheir-Neiss et al., Appl.Microbiol. Biotechnology 20:46-53, 1984, which is hereby incorporated byreference in its entirety, particularly with respect to strains of T.reesei.

In some embodiments, the source organism is a yeast, such asSaccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp.

In some embodiments, the source organism is a bacterium, such as strainsof Bacillus such as B. lichenformis or B. subtilis, strains of Pantoeasuch as P. citrea, strains of Pseudomonas such as P. alcaligenes,strains of Streptomyces such as S. lividans or S. rubiginosus, orstrains of Escherichia such as E. coli.

As used herein, “the genus Bacillus” includes all species within thegenus “Bacillus,” as known to those of skill in the art, including butnot limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii,B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, andB. thuringiensis. It is recognized that the genus Bacillus continues toundergo taxonomical reorganization. Thus, it is intended that the genusinclude species that have been reclassified, including but not limitedto such organisms as B. stearothermophilus, which is now named“Geobacillus stearothermophilus.” The production of resistant endosporesin the presence of oxygen is considered the defining feature of thegenus Bacillus, although this characteristic also applies to therecently named Alicyclobacillus, Amphibacillus, Aneurinibacillus,Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus,and Virgibacillus.

In some embodiments, the source organism is a gram-positive bacterium.Non-limiting examples include strains of Streptomyces (e.g., S.lividans, S. coelicolor, or S. griseus) and Bacillus. In someembodiments, the source organism is a gram-negative bacterium, such asE. coli or Pseudomonas sp.

In some embodiments, the source organism is a plant, such as a plantfrom the family Fabaceae, such as the Faboideae subfamily. In someembodiments, the source organism is kudzu, poplar (such as Populusalba×tremula CAC35696), aspen (such as Populus tremuloides), or Quercusrobur.

In some embodiments, the source organism is an algae, such as a greenalgae, red algae, glaucophytes, chlorarachniophytes, euglenids,chromista, or dinoflagellates.

In some embodiments, the source organism is a cyanobacteria, such ascyanobacteria classified into any of the following groups based onmorphology: Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales,or Stigonematales.

Exemplary Host Cells

A variety of host cells can be used to express isoprene synthase, DXS,IDI, and/or MVA pathway polypeptides and to produce isoprene in themethods of the claimed invention. Exemplary host cells include cellsfrom any of the organisms listed in the prior section under the heading“Exemplary Source Organisms.” The host cell may be a cell that naturallyproduces isoprene or a cell that does not naturally produce isoprene. Insome embodiments, the host cell naturally produces isoprene using theDXP pathway, and an isoprene synthase, DXS, and/or IDI nucleic acid isadded to enhance production of isoprene using this pathway. In someembodiments, the host cell naturally produces isoprene using the MVApathway, and an isoprene synthase and/or one or more MVA pathway nucleicacids are added to enhance production of isoprene using this pathway. Insome embodiments, the host cell naturally produces isoprene using theDXP pathway and one or more MVA pathway nucleic acids are added toproduce isoprene using part or all of the MVA pathway as well as the DXPpathway. In some embodiments, the host cell naturally produces isopreneusing both the DXP and MVA pathways and one or more isoprene synthase,DXS, IDI, or MVA pathway nucleic acids are added to enhance productionof isoprene by one or both of these pathways.

Exemplary Transformation Methods

Isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids or vectorscontaining them can be inserted into a host cell (e.g., a plant cell, afungal cell, a yeast cell, or a bacterial cell described herein) usingstandard techniques for expression of the encoded isoprene synthase,DXS, IDI, and/or MVA pathway polypeptide. Introduction of a DNAconstruct or vector into a host cell can be performed using techniquessuch as transformation, electroporation, nuclear microinjection,transduction, transfection (e.g., lipofection mediated or DEAE-Dextrinmediated transfection or transfection using a recombinant phage virus),incubation with calcium phosphate DNA precipitate, high velocitybombardment with DNA-coated microprojectiles, and protoplast fusion.General transformation techniques are known in the art (see, e.g.,Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds)Chapter 9, 1987; Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor, 1989; and Campbell et al., Curr.Genet. 16:53-56, 1989, which are each hereby incorporated by referencein their entireties, particularly with respect to transformationmethods). The expression of heterologous polypeptide in Trichoderma isdescribed in U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328; U.S. Pat.No. 7,262,041; WO 2005/001036; Harkki et al.; Enzyme Microb. Technol.13:227-233, 1991; Harkki et al., Bio Technol. 7:596-603, 1989; EP244,234; EP 215,594; and Nevalainen et al., “The Molecular Biology ofTrichoderma and its Application to the Expression of Both Homologous andHeterologous Genes,” in Molecular Industrial Mycology, Eds. Leong andBerka, Marcel Dekker Inc., NY pp. 129-148, 1992, which are each herebyincorporated by reference in their entireties, particularly with respectto transformation and expression methods). Reference is also made to Caoet al., (Sci. 9:991-1001, 2000; EP 238023; and Yelton et al.,Proceedings. Natl. Acad. Sci. USA 81:1470-1474, 1984 (which are eachhereby incorporated by reference in their entireties, particularly withrespect to transformation methods) for transformation of Aspergillusstrains. The introduced nucleic acids may be integrated into chromosomalDNA or maintained as extrachromosomal replicating sequences.

Any method known in the art may be used to select transformants. In onenon-limiting example, stable transformants including an amdS marker aredistinguished from unstable transformants by their faster growth rateand the formation of circular colonies with a smooth, rather than raggedoutline on solid culture medium containing acetamide. Additionally, insome cases a further test of stability is conducted by growing thetransformants on a solid non-selective medium (e.g., a medium that lacksacetamide), harvesting spores from this culture medium, and determiningthe percentage of these spores which subsequently germinate and grow onselective medium containing acetamide.

In some embodiments, fungal cells are transformed by a process involvingprotoplast formation and transformation of the protoplasts followed byregeneration of the cell wall in a known manner. In one specificembodiment, the preparation of Trichoderma sp. for transformationinvolves the preparation of protoplasts from fungal mycelia (see,Campbell et al., Curr. Genet. 16:53-56, 1989, which is incorporated byreference in its entirety, particularly with respect to transformationmethods). In some embodiments, the mycelia are obtained from germinatedvegetative spores. The mycelia are treated with an enzyme that digeststhe cell wall resulting in protoplasts. The protoplasts are thenprotected by the presence of an osmotic stabilizer in the suspendingmedium. These stabilizers include sorbitol, mannitol, potassiumchloride, magnesium sulfate, and the like. Usually the concentration ofthese stabilizers varies between 0.8 M and 1.2 M. It is desirable to useabout a 1.2 M solution of sorbitol in the suspension medium.

Uptake of DNA into the host Trichoderma sp. strain is dependent upon thecalcium ion concentration. Generally, between about 10 mM CaCl₂ and 50mM CaCl₂ is used in an uptake solution. In addition to the calcium ionin the uptake solution, other compounds generally included are abuffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethyleneglycol (PEG). While not intending to be bound to any particular theory,it is believed that the polyethylene glycol acts to fuse the cellmembranes, thus permitting the contents of the medium to be deliveredinto the cytoplasm of the Trichoderma sp. strain and the plasmid DNA tobe transferred to the nucleus. This fusion frequently leaves multiplecopies of the plasmid DNA integrated into the host chromosome.

Usually a suspension containing the Trichoderma sp. protoplasts or cellsthat have been subjected to a permeability treatment at a density of 10⁵to 10⁷/mL (such as 2×10⁶/mL) are used in the transformation. A volume of100 μL of these protoplasts or cells in an appropriate solution (e.g.,1.2 M sorbitol and 50 mM CaCl₂) are mixed with the desired DNA.Generally, a high concentration of PEG is added to the uptake solution.From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplastsuspension. In some embodiments, about 0.25 volumes are added to theprotoplast suspension. Additives such as dimethyl sulfoxide, heparin,spermidine, potassium chloride, and the like may also be added to theuptake solution and aid in transformation. Similar procedures areavailable for other fungal host cells (see, e.g., U.S. Pat. Nos.6,022,725 and 6,268,328, which are each hereby incorporated by referencein their entireties, particularly with respect to transformationmethods).

Generally, the mixture is then cultured at approximately 0° C. for aperiod of between 10 to 30 minutes. Additional PEG is then added to themixture to further enhance the uptake of the desired nucleic acidsequence. The 25% PEG 4000 is generally added in volumes of 5 to 15times the volume of the transformation mixture; however, greater andlesser volumes may be suitable. The 25% PEG 4000 is desirably about 10times the volume of the transformation mixture. After the PEG is added,the transformation mixture is then cultured either at room temperatureor on ice before the addition of a sorbitol and CaCl₂ solution. Theprotoplast suspension is then further added to molten aliquots of agrowth medium. When the growth medium includes a growth selection (e.g.,acetamide or an antibiotic) it permits the growth of transformants only.

The transformation of bacterial cells may be performed according toconventional methods, e.g., as described in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, 1982, which is herebyincorporated by reference in its entirety, particularly with respect totransformation methods.

Exemplary Cell Culture Media

The invention also includes a cell or a population of cells in culturethat produce isoprene. By “cells in culture” is meant two or more cellsin a solution (e.g., a cell medium) that allows the cells to undergo oneor more cell divisions. “Cells in culture” do not include plant cellsthat are part of a living, multicellular plant containing cells thathave differentiated into plant tissues. In various embodiments, the cellculture includes at least or about 10, 20, 50, 100, 200, 500, 1,000,5,000, 10,000 or more cells.

Any carbon source can be used to cultivate the host cells. The term“carbon source” refers to one or more carbon-containing compoundscapable of being metabolized by a host cell or organism. For example,the cell medium used to cultivate the host cells may include any carbonsource suitable for maintaining the viability or growing the host cells.

In some embodiments, the carbon source is a carbohydrate (such asmonosaccharide, disaccharide, oligosaccharide, or polysaccharids),invert sugar (e.g., enzymatically treated sucrose syrup), glycerol,glycerine (e.g., a glycerine byproduct of a biodiesel or soap-makingprocess), dihydroxyacetone, one-carbon source, oil (e.g., a plant orvegetable oil such as corn, palm, or soybean oil), animal fat, animaloil, fatty acid (e.g., a saturated fatty acid, unsaturated fatty acid,or polyunsaturated fatty acid), lipid, phospholipid, glycerolipid,monoglyceride, diglyceride, triglyceride, polypeptide (e.g., a microbialor plant protein or peptide), renewable carbon source (e.g., a biomasscarbon source such as a hydrolyzed biomass carbon source), yeastextract, component from a yeast extract, polymer, acid, alcohol,aldehyde, ketone, amino acid, succinate, lactate, acetate, ethanol, orany combination of two or more of the foregoing. In some embodiments,the carbon source is a product of photosynthesis, including, but notlimited to, glucose.

Exemplary monosaccharides include glucose and fructose; exemplaryoligosaccharides include lactose and sucrose, and exemplarypolysaccharides include starch and cellulose. Exemplary carbohydratesinclude C6 sugars (e.g., fructose, mannose, galactose, or glucose) andC5 sugars (e.g., xylose or arabinose). In some embodiments, the cellmedium includes a carbohydrate as well as a carbon source other than acarbohydrate (e.g., glycerol, glycerine, dihydroxyacetone, one-carbonsource, oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, or a component from a yeast extract). In some embodiments, thecell medium includes a carbohydrate as well as a polypeptide (e.g., amicrobial or plant protein or peptide). In some embodiments, themicrobial polypeptide is a polypeptide from yeast or bacteria. In someembodiments, the plant polypeptide is a polypeptide from soy, corn,canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed,cottonseed, palm kernel, olive, safflower, sesame, or linseed.

In some embodiments, the concentration of the carbohydrate is at leastor about 5 grams per liter of broth (g/L, wherein the volume of brothincludes both the volume of the cell medium and the volume of thecells), such as at least or about 10, 15, 20, 30, 40, 50, 60, 80, 100,150, 200, 300, 400, or more g/L. In some embodiments, the concentrationof the carbohydrate is between about 50 and about 400 g/L, such asbetween about 100 and about 360 g/L, between about 120 and about 360g/L, or between about 200 and about 300 g/L. In some embodiments, thisconcentration of carbohydrate includes the total amount of carbohydratethat is added before and/or during the culturing of the host cells.

In some embodiments, the cells are cultured under limited glucoseconditions. By “limited glucose conditions” is meant that the amount ofglucose that is added is less than or about 105% (such as about 100%) ofthe amount of glucose that is consumed by the cells. In particularembodiments, the amount of glucose that is added to the culture mediumis approximately the same as the amount of glucose that is consumed bythe cells during a specific period of time. In some embodiments, therate of cell growth is controlled by limiting the amount of addedglucose such that the cells grow at the rate that can be supported bythe amount of glucose in the cell medium. In some embodiments, glucosedoes not accumulate during the time the cells are cultured. In variousembodiments, the cells are cultured under limited glucose conditions forgreater than or about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, or70 hours. In various embodiments, the cells are cultured under limitedglucose conditions for greater than or about 5, 10, 15, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 95, or 100% of the total length of time thecells are cultured. While not intending to be bound by any particulartheory, it is believed that limited glucose conditions may allow morefavorable regulation of the cells.

In some embodiments, the cells are cultured in the presence of an excessof glucose. In particular embodiments, the amount of glucose that isadded is greater than about 105% (such as about or greater than 110,120, 150, 175, 200, 250, 300, 400, or 500%) or more of the amount ofglucose that is consumed by the cells during a specific period of time.In some embodiments, glucose accumulates during the time the cells arecultured.

Exemplary lipids are any substance containing one or more fatty acidsthat are C4 and above fatty acids that are saturated, unsaturated, orbranched.

Exemplary oils are lipids that are liquid at room temperature. In someembodiments, the lipid contains one or more C4 or above fatty acids(e.g., contains one or more saturated, unsaturated, or branched fattyacid with four or more carbons). In some embodiments, the oil isobtained from soy, corn, canola, jatropha, palm, peanut, sunflower,coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower,sesame, linseed, oleagineous microbial cells, Chinese tallow, or anycombination of two or more of the foregoing.

Exemplary fatty acids include compounds of the formula RCOOH, where “R”is a hydrocarbon. Exemplary unsaturated fatty acids include compoundswhere “R” includes at least one carbon-carbon double bond. Exemplaryunsaturated fatty acids include, but are not limited to, oleic acid,vaccenic acid, linoleic acid, palmitelaidic acid, and arachidonic acid.Exemplary polyunsaturated fatty acids include compounds where “R”includes a plurality of carbon-carbon double bonds. Exemplary saturatedfatty acids include compounds where “R” is a saturated aliphatic group.In some embodiments, the carbon source includes one or more C₁₂-C₂₂fatty acids, such as a C₁₂ saturated fatty acid, a C₁₄ saturated fattyacid, a C₁₆ saturated fatty acid, a C₁₈ saturated fatty acid, a C₂₀saturated fatty acid, or a C₂₂ saturated fatty acid. In an exemplaryembodiment, the fatty acid is palmitic acid. In some embodiments, thecarbon source is a salt of a fatty acid (e.g., an unsaturated fattyacid), a derivative of a fatty acid (e.g., an unsaturated fatty acid),or a salt of a derivative of fatty acid (e.g., an unsaturated fattyacid). Suitable salts include, but are not limited to, lithium salts,potassium salts, sodium salts, and the like. Di- and triglycerols arefatty acid esters of glycerol.

In some embodiments, the concentration of the lipid, oil, fat, fattyacid, monoglyceride, diglyceride, or triglyceride is at least or about 1gram per liter of broth (g/L, wherein the volume of broth includes boththe volume of the cell medium and the volume of the cells), such as atleast or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300,400, or more g/L. In some embodiments, the concentration of the lipid,oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride isbetween about 10 and about 400 g/L, such as between about 25 and about300 g/L, between about 60 and about 180 g/L, or between about 75 andabout 150 g/L. In some embodiments, the concentration includes the totalamount of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride,or triglyceride that is added before and/or during the culturing of thehost cells. In some embodiments, the carbon source includes both (i) alipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglycerideand (ii) a carbohydrate, such as glucose. In some embodiments, the ratioof the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, ortriglyceride to the carbohydrate is about 1:1 on a carbon basis (i.e.,one carbon in the lipid, oil, fat, fatty acid, monoglyceride,diglyceride, or triglyceride per carbohydrate carbon). In particularembodiments, the amount of the lipid, oil, fat, fatty acid,monoglyceride, diglyceride, or triglyceride is between about 60 and 180g/L, and the amount of the carbohydrate is between about 120 and 360g/L.

Exemplary microbial polypeptide carbon sources include one or morepolypeptides from yeast or bacteria. Exemplary plant polypeptide carbonsources include one or more polypeptides from soy, corn, canola,jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed,cottonseed, palm kernel, olive, safflower, sesame, or linseed.

Exemplary renewable carbon sources include cheese whey permeate,cornsteep liquor, sugar beet molasses, barley malt, and components fromany of the foregoing. Exemplary renewable carbon sources also includeglucose, hexose, pentose and xylose present in biomass, such as corn,switchgrass, sugar cane, cell waste of fermentation processes, andprotein by-product from the milling of soy, corn, or wheat. In someembodiments, the biomass carbon source is a lignocellulosic,hemicellulosic, or cellulosic material such as, but are not limited to,a grass, wheat, wheat straw, bagasse, sugar cane bagasse, soft woodpulp, corn, corn cob or husk, corn kernel, fiber from corn kernels, cornstover, switch grass, rice hull product, or a by-product from wet or drymilling of grains (e.g., corn, sorghum, rye, triticate, barley, wheat,and/or distillers grains). Exemplary cellulosic materials include wood,paper and pulp waste, herbaceous plants, and fruit pulp. In someembodiments, the carbon source includes any plant part, such as stems,grains, roots, or tubers. In some embodiments, all or part of any of thefollowing plants are used as a carbon source: corn, wheat, rye, sorghum,triticate, rice, millet, barley, cassaya, legumes, such as beans andpeas, potatoes, sweet potatoes, bananas, sugarcane, and/or tapioca. Insome embodiments, the carbon source is a biomass hydrolysate, such as abiomass hydrolysate that includes both xylose and glucose or thatincludes both sucrose and glucose.

In some embodiments, the renewable carbon source (such as biomass) ispretreated before it is added to the cell culture medium. In someembodiments, the pretreatment includes enzymatic pretreatment, chemicalpretreatment, or a combination of both enzymatic and chemicalpretreatment (see, for example, Farzaneh et al., Bioresource Technology96 (18): 2014-2018, 2005; U.S. Pat. No. 6,176,176; U.S. Pat. No.6,106,888; which are each hereby incorporated by reference in theirentireties, particularly with respect to the pretreatment of renewablecarbon sources). In some embodiments, the renewable carbon source ispartially or completely hydrolyzed before it is added to the cellculture medium.

In some embodiments, the renewable carbon source (such as corn stover)undergoes ammonia fiber expansion (AFEX) pretreatment before it is addedto the cell culture medium (see, for example, Farzaneh et al.,Bioresource Technology 96 (18): 2014-2018, 2005). During AFEXpretreatment, a renewable carbon source is treated with liquid anhydrousammonia at moderate temperatures (such as about 60 to about 100° C.) andhigh pressure (such as about 250 to about 300 psi) for about 5 minutes.Then, the pressure is rapidly released. In this process, the combinedchemical and physical effects of lignin solubilization, hemicellulosehydrolysis, cellulose decrystallization, and increased surface areaenables near complete enzymatic conversion of cellulose andhemicellulose to fermentable sugars. AFEX pretreatment has the advantagethat nearly all of the ammonia can be recovered and reused, while theremaining serves as nitrogen source for microbes in downstreamprocesses. Also, a wash stream is not required for AFEX pretreatment.Thus, dry matter recovery following the AFEX treatment is essentially100%. AFEX is basically a dry to dry process. The treated renewablecarbon source is stable for long periods and can be fed at very highsolid loadings in enzymatic hydrolysis or fermentation processes.Cellulose and hemicellulose are well preserved in the AFEX process, withlittle or no degradation. There is no need for neutralization prior tothe enzymatic hydrolysis of a renewable carbon source that has undergoneAFEX pretreatment. Enzymatic hydrolysis of AFEX-treated carbon sourcesproduces clean sugar streams for subsequent fermentation use.

In some embodiments, the concentration of the carbon source (e.g., arenewable carbon source) is equivalent to at least or about 0.1, 0.5, 1,1.5 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50% glucose (w/v). The equivalentamount of glucose can be determined by using standard HPLC methods withglucose as a reference to measure the amount of glucose generated fromthe carbon source. In some embodiments, the concentration of the carbonsource (e.g., a renewable carbon source) is equivalent to between about0.1 and about 20% glucose, such as between about 0.1 and about 10%glucose, between about 0.5 and about 10% glucose, between about 1 andabout 10% glucose, between about 1 and about 5% glucose, or betweenabout 1 and about 2% glucose.

In some embodiments, the carbon source includes yeast extract or one ormore components of yeast extract. In some embodiments, the concentrationof yeast extract is at least 1 gram of yeast extract per liter of broth(g/L, wherein the volume of broth includes both the volume of the cellmedium and the volume of the cells), such at least or about 5, 10, 15,20, 30, 40, 50, 60, 80, 100, 150, 200, 300, or more g/L. In someembodiments, the concentration of yeast extract is between about 1 andabout 300 g/L, such as between about 1 and about 200 g/L, between about5 and about 200 g/L, between about 5 and about 100 g/L, or between about5 and about 60 g/L. In some embodiments, the concentration includes thetotal amount of yeast extract that is added before and/or during theculturing of the host cells. In some embodiments, the carbon sourceincludes both yeast extract (or one or more components thereof) andanother carbon source, such as glucose. In some embodiments, the ratioof yeast extract to the other carbon source is about 1:5, about 1:10, orabout 1:20 (w/w).

Additionally the carbon source may also be one-carbon substrates such ascarbon dioxide, or methanol. Glycerol production from single carbonsources (e.g., methanol, formaldehyde, or formate) has been reported inmethylotrophic yeasts (Yamada et al., Agric. Biol. Chem., 53(2) 541-543,1989, which is hereby incorporated by reference in its entirety,particularly with respect to carbon sources) and in bacteria (Hunter et.al., Biochemistry, 24, 4148-4155, 1985, which is hereby incorporated byreference in its entirety, particularly with respect to carbon sources).These organisms can assimilate single carbon compounds, ranging inoxidation state from methane to formate, and produce glycerol. Thepathway of carbon assimilation can be through ribulose monophosphate,through serine, or through xylulose-momophosphate (Gottschalk, BacterialMetabolism, Second Edition, Springer-Verlag: New York, 1986, which ishereby incorporated by reference in its entirety, particularly withrespect to carbon sources). The ribulose monophosphate pathway involvesthe condensation of formate with ribulose-5-phosphate to form a sixcarbon sugar that becomes fructose and eventually the three carbonproduct glyceraldehyde-3-phosphate. Likewise, the serine pathwayassimilates the one-carbon compound into the glycolytic pathway viamethylenetetrahydrofolate.

In addition to one and two carbon substrates, methylotrophic organismsare also known to utilize a number of other carbon containing compoundssuch as methylamine, glucosamine and a variety of amino acids formetabolic activity. For example, methylotrophic yeast are known toutilize the carbon from methylamine to form trehalose or glycerol(Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7^(th) ed.,415-32. Editors: Murrell et al., Publisher: Intercept, Andover, UK,1993, which is hereby incorporated by reference in its entirety,particularly with respect to carbon sources). Similarly, various speciesof Candida metabolize alanine or oleic acid (Sulter et al., Arch.Microbiol. 153(5), 485-9, 1990, which is hereby incorporated byreference in its entirety, particularly with respect to carbon sources).

In some embodiments, cells are cultured in a standard medium containingphysiological salts and nutrients (see, e.g., Pourquie, J. et al.,Biochemistry and Genetics of Cellulose Degradation, eds. Aubert et al.,Academic Press, pp. 71-86, 1988 and Ilmen et al., Appl. Environ.Microbiol. 63:1298-1306, 1997, which are each hereby incorporated byreference in their entireties, particularly with respect to cellmedias). Exemplary growth media are common commercially prepared mediasuch as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, orYeast medium (YM) broth. Other defined or synthetic growth media mayalso be used, and the appropriate medium for growth of particular hostcells are known by someone skilled in the art of microbiology orfermentation science.

In addition to an appropriate carbon source, the cell medium desirablycontains suitable minerals, salts, cofactors, buffers, and othercomponents known to those skilled in the art suitable for the growth ofthe cultures or the enhancement of isoprene production (see, forexample, WO 2004/033646 and references cited therein and WO 96/35796 andreferences cited therein, which are each hereby incorporated byreference in their entireties, particularly with respect cell medias andcell culture conditions). In some embodiments where an isoprenesynthase, DXS, IDI, and/or MVA pathway nucleic acid is under the controlof an inducible promoter, the inducing agent (e.g., a sugar, metal saltor antimicrobial), is desirably added to the medium at a concentrationeffective to induce expression of an isoprene synthase, DXS, IDI, and/orMVA pathway polypeptide. In some embodiments, cell medium has anantibiotic (such as kanamycin) that corresponds to the antibioticresistance nucleic acid (such as a kanamycin resistance nucleic acid) ona vector that has one or more DXS, IDI, or MVA pathway nucleic acids.

Exemplary Cell Culture Conditions

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Exemplary techniques maybe found in Manual of Methods for General Bacteriology Gerhardt et al.,eds), American Society for Microbiology, Washington, D.C. (1994) orBrock in Biotechnology: A Textbook of Industrial Microbiology, SecondEdition (1989) Sinauer Associates, Inc., Sunderland, Mass., which areeach hereby incorporated by reference in their entireties, particularlywith respect to cell culture techniques. In some embodiments, the cellsare cultured in a culture medium under conditions permitting theexpression of one or more isoprene synthase, DXS, IDI, or MVA pathwaypolypeptides encoded by a nucleic acid inserted into the host cells.

Standard cell culture conditions can be used to culture the cells (see,for example, WO 2004/033646 and references cited therein, which are eachhereby incorporated by reference in their entireties, particularly withrespect to cell culture and fermentation conditions). Cells are grownand maintained at an appropriate temperature, gas mixture, and pH (suchas at about 20 to about 37° C., at about 6% to about 84% CO₂, and at apH between about 5 to about 9). In some embodiments, cells are grown at35° C. in an appropriate cell medium. In some embodiments, e.g.,cultures are cultured at approximately 28° C. in appropriate medium inshake cultures or fermentors until desired amount of isoprene productionis achieved. In some embodiments, the pH ranges for fermentation arebetween about pH 5.0 to about pH 9.0 (such as about pH 6.0 to about pH8.0 or about 6.5 to about 7.0). Reactions may be performed underaerobic, anoxic, or anaerobic conditions based on the requirements ofthe host cells. Exemplary culture conditions for a given filamentousfungus are known in the art and may be found in the scientificliterature and/or from the source of the fungi such as the American TypeCulture Collection and Fungal Genetics Stock Center.

In various embodiments, the cells are grown using any known mode offermentation, such as batch, fed-batch, or continuous processes. In someembodiments, a batch method of fermentation is used. Classical batchfermentation is a closed system where the composition of the media isset at the beginning of the fermentation and is not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the cell medium is inoculated with the desired hostcells and fermentation is permitted to occur adding nothing to thesystem. Typically, however, “batch” fermentation is batch with respectto the addition of carbon source and attempts are often made atcontrolling factors such as pH and oxygen concentration. In batchsystems, the metabolite and biomass compositions of the system changeconstantly until the time the fermentation is stopped. Within batchcultures, cells moderate through a static lag phase to a high growth logphase and finally to a stationary phase where growth rate is diminishedor halted. In some embodiments, cells in log phase are responsible forthe bulk of the isoprene production. In some embodiments, cells instationary phase produce isoprene.

In some embodiments, a variation on the standard batch system is used,such as the Fed-Batch system. Fed-Batch fermentation processes comprisea typical batch system with the exception that the carbon source isadded in increments as the fermentation progresses. Fed-Batch systemsare useful when catabolite repression is apt to inhibit the metabolismof the cells and where it is desirable to have limited amounts of carbonsource in the cell medium. Fed-batch fermentations may be performed withthe carbon source (e.g., glucose) in a limited or excess amount.Measurement of the actual carbon source concentration in Fed-Batchsystems is difficult and is therefore estimated on the basis of thechanges of measurable factors such as pH, dissolved oxygen, and thepartial pressure of waste gases such as CO₂. Batch and Fed-Batchfermentations are common and well known in the art and examples may befound in Brock, Biotechnology: A Textbook of Industrial Microbiology,Second Edition (1989) Sinauer Associates, Inc., which is herebyincorporated by reference in its entirety, particularly with respect tocell culture and fermentation conditions.

In some embodiments, continuous fermentation methods are used.Continuous fermentation is an open system where a defined fermentationmedium is added continuously to a bioreactor and an equal amount ofconditioned medium is removed simultaneously for processing. Continuousfermentation generally maintains the cultures at a constant high densitywhere cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or isoprene production. Forexample, one method maintains a limiting nutrient such as the carbonsource or nitrogen level at a fixed rate and allows all other parametersto moderate. In other systems, a number of factors affecting growth canbe altered continuously while the cell concentration (e.g., theconcentration measured by media turbidity) is kept constant. Continuoussystems strive to maintain steady state growth conditions. Thus, thecell loss due to media being drawn off is balanced against the cellgrowth rate in the fermentation. Methods of modulating nutrients andgrowth factors for continuous fermentation processes as well astechniques for maximizing the rate of product formation are well knownin the art of industrial microbiology and a variety of methods aredetailed by Brock, Biotechnology: A Textbook of Industrial Microbiology,Second Edition (1989) Sinauer Associates, Inc., which is herebyincorporated by reference in its entirety, particularly with respect tocell culture and fermentation conditions.

In some embodiments, cells are immobilized on a substrate as whole cellcatalysts and subjected to fermentation conditions for isopreneproduction.

In some embodiments, bottles of liquid culture are placed in shakers inorder to introduce oxygen to the liquid and maintain the uniformity ofthe culture. In some embodiments, an incubator is used to control thetemperature, humidity, shake speed, and/or other conditions in which aculture is grown. The simplest incubators are insulated boxes with anadjustable heater, typically going up to ˜65° C. More elaborateincubators can also include the ability to lower the temperature (viarefrigeration), or the ability to control humidity or CO₂ levels. Mostincubators include a timer; some can also be programmed to cycle throughdifferent temperatures, humidity levels, etc. Incubators can vary insize from tabletop to units the size of small rooms.

If desired, a portion or all of the cell medium can be changed toreplenish nutrients and/or avoid the build up of potentially harmfulmetabolic byproducts and dead cells. In the case of suspension cultures,cells can be separated from the media by centrifuging or filtering thesuspension culture and then resuspending the cells in fresh media. Inthe case of adherent cultures, the media can be removed directly byaspiration and replaced. In some embodiments, the cell medium allows atleast a portion of the cells to divide for at least or about 5, 10, 20,40, 50, 60, 65, or more cell divisions in a continuous culture (such asa continuous culture without dilution).

In some embodiments, a constitutive or leaky promoter (such as a Trcpromoter) is used and a compound (such as IPTG) is not added to induceexpression of the isoprene synthase, DXS, IDI, or MVA pathway nucleicacid(s) operably linked to the promoter. In some embodiments, a compound(such as IPTG) is added to induce expression of the isoprene synthase,DXS, IDI, or MVA pathway nucleic acid(s) operably linked to thepromoter.

Exemplary Methods for Decoupling Isoprene Production from Cell Growth

Desirably, carbon from the feedstock is converted to isoprene ratherthan to the growth and maintenance of the cells. In some embodiments,the cells are grown to a low to medium OD₆₀₀, then production ofisoprene is started or increased. This strategy permits a large portionof the carbon to be converted to isoprene.

In some embodiments, cells reach an optical density such that they nolonger divide or divide extremely slowly, but continue to make isoprenefor several hours (such as about 2, 4, 6, 8, 10, 15, 20, 25, 30, or morehours). For example, FIGS. 60A-67C illustrate that cells may continue toproduce a substantial amount of mevalonic acid or isoprene after thecells reach an optical density such that they no longer divide or divideextremely slowly. In some cases, the optical density at 550 nm decreasesover time (such as a decrease in the optical density after the cells areno longer in an exponential growth phase due to cell lysis, cessation ofgrowth, lack of nutrients or other factors leading to lack of cellgrowth), and the cells continue to produce a substantial amount ofmevalonic acid or isoprene. In some embodiments, the optical density at550 nm of the cells increases by less than or about 50% (such as by lessthan or about 40, 30, 20, 10, 5, or 0%) over a certain time period (suchas greater than or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), andthe cells produce isoprene at greater than or about 1, 10, 25, 50, 100,150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000; 1,250; 1,500;1,750; 2,000; 2,500; 3,000; 4,000; 5,000; 10,000; 20,000; 30,000;40,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000;700,000; 800,000; 900,000; 1,000,000 or more nmole of isoprene/gram ofcells for the wet weight of the cells/hour (nmole/g_(wcm)/hr) duringthis time period. In some embodiments, the amount of isoprene is betweenabout 2 to about 5,000 nmole/g_(wcm)/hr, such as between about 2 toabout 100 nmole/g_(wcm)/hr, about 100 to about 500 nmole/g_(wcm)/hr,about 150 to about 500 nmole/g_(wcm)/hr, about 500 to about 1,000nmole/g_(wcm)/hr, about 1,000 to about 2,000 nmole/g_(wcm)/hr, or about2,000 to about 5,000 nmole/g_(wcm)/hr. In some embodiments, the amountof isoprene is between about 20 to about 5,000 nmole/g_(wcm)/hr, about100 to about 5,000 nmole/g_(wcm)/hr, about 200 to about 2,000nmole/g_(wcm)/hr, about 200 to about 1,000 nmole/g_(wcm)/hr, about 300to about 1,000 nmole/g_(wcm)/hr, or about 400 to about 1,000nmole/g_(wcm)/hr.

In some embodiments, the optical density at 550 nm of the cellsincreases by less than or about 50% (such as by less than or about 40,30, 20, 10, 5, or 0%) over a certain time period (such as greater thanor about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cellsproduce a cumulative titer (total amount) of isoprene at greater than orabout 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800,900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000,10,000, 50,000, 100,000, or more mg of isoprene/L of broth(mg/L_(broth), wherein the volume of broth includes the volume of thecells and the cell medium) during this time period. In some embodiments,the amount of isoprene is between about 2 to about 5,000 mg/L_(broth),such as between about 2 to about 100 mg/L_(broth), about 100 to about500 mg/L_(broth), about 500 to about 1,000 mg/L_(broth), about 1,000 toabout 2,000 mg/L_(broth), or about 2,000 to about 5,000 mg/L_(broth). Insome embodiments, the amount of isoprene is between about 20 to about5,000 mg/L_(broth), about 100 to about 5,000 mg/L_(broth), about 200 toabout 2,000 mg/L_(broth), about 200 to about 1,000 mg/L_(broth), about300 to about 1,000 mg/L_(broth), or about 400 to about 1,000mg/L_(broth).

In some embodiments, the optical density at 550 nm of the cellsincreases by less than or about 50% (such as by less than or about 40,30, 20, 10, 5, or 0%) over a certain time period (such as greater thanor about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cellsconvert greater than or about 0.0015, 0.002, 0.005, 0.01, 0.02, 0.05,0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2,1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, or 8.0% of thecarbon in the cell culture medium into isoprene during this time period.In some embodiments, the percent conversion of carbon into isoprene isbetween such as about 0.002 to about 4.0%, about 0.002 to about 3.0%,about 0.002 to about 2.0%, about 0.002 to about 1.6%, about 0.002 toabout 0.005%, about 0.005 to about 0.01%, about 0.01 to about 0.05%,about 0.05 to about 0.15%, 0.15 to about 0.2%, about 0.2 to about 0.3%,about 0.3 to about 0.5%, about 0.5 to about 0.8%, about 0.8 to about1.0%, or about 1.0 to about 1.6%. In some embodiments, the percentconversion of carbon into isoprene is between about 0.002 to about 0.4%,0.002 to about 0.16%, 0.04 to about 0.16%, about 0.005 to about 0.3%,about 0.01 to about 0.3%, or about 0.05 to about 0.3%.

In some embodiments, isoprene is only produced in stationary phase. Insome embodiments, isoprene is produced in both the growth phase andstationary phase. In various embodiments, the amount of isopreneproduced (such as the total amount of isoprene produced or the amount ofisoprene produced per liter of broth per hour per OD₆₀₀) duringstationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40,50, or more times the amount of isoprene produced during the growthphase for the same length of time. In various embodiments, greater thanor about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99% or more of thetotal amount of isoprene that is produced (such as the production ofisoprene during a fermentation for a certain amount of time, such as 20hours) is produced while the cells are in stationary phase. In variousembodiments, greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80,90, 95, 99% or more of the total amount of isoprene that is produced(such as the production of isoprene during a fermentation for a certainamount of time, such as 20 hours) is produced while the cells divideslowly or not at all such that the optical density at 550 nm of thecells increases by less than or about 50% (such as by less than or about40, 30, 20, 10, 5, or 0%). In some embodiments, isoprene is onlyproduced in the growth phase.

In some embodiments, one or more MVA pathway, IDI, DXP, or isoprenesynthase nucleic acids are placed under the control of a promoter orfactor that is more active in stationary phase than in the growth phase.For example, one or more MVA pathway, IDI, DXP, or isoprene synthasenucleic acids may be placed under control of a stationary phase sigmafactor, such as RpoS. In some embodiments, one or more MVA pathway, IDI,DXP, or isoprene synthase nucleic acids are placed under control of apromoter inducible in stationary phase, such as a promoter inducible bya response regulator active in stationary phase.

Production of Isoprene within Safe Operating Ranges

The production of isoprene within safe operating levels according to itsflammability characteristics simplifies the design and construction ofcommercial facilities, vastly improves the ability to operate safely,and limits the potential for fires to occur. In particular, the optimalranges for the production of isoprene are within the safe zone, i.e.,the nonflammable range of isoprene concentrations. In one such aspect,the invention features a method for the production of isoprene withinthe nonflammable range of isoprene concentrations (outside theflammability envelope of isoprene).

Thus, computer modeling and experimental testing were used to determinethe flammability limits of isoprene (such as isoprene in the presence ofO₂, N₂, CO₂, or any combination of two or more of the foregoing gases)in order to ensure process safety. The flammability envelope ischaracterized by the lower flammability limit (LFL), the upperflammability limit (UFL), the limiting oxygen concentration (LOC), andthe limiting temperature. For a system to be flammable, a minimum amountof fuel (such as isoprene) must be in the presence of a minimum amountof oxidant, typically oxygen. The LFL is the minimum amount of isoprenethat must be present to sustain burning, while the UFL is the maximumamount of isoprene that can be present. Above this limit, the mixture isfuel rich and the fraction of oxygen is too low to have a flammablemixture. The LOC indicates the minimum fraction of oxygen that must alsobe present to have a flammable mixture. The limiting temperature isbased on the flash point of isoprene and is that lowest temperature atwhich combustion of isoprene can propagate. These limits are specific tothe concentration of isoprene, type and concentration of oxidant, inertspresent in the system, temperature, and pressure of the system.Compositions that fall within the limits of the flammability envelopepropagate combustion and require additional safety precautions in boththe design and operation of process equipment.

The following conditions were tested using computer simulation andmathematical analysis and experimental testing. If desired, otherconditions (such as other temperature, pressure, and permanent gascompositions) may be tested using the methods described herein todetermine the LFL, UFL, and LOC concentrations.

(1) Computer simulation and mathematical analysis

Test Suite 1:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt % N₂: 79 wt %-94 wt % Test Suite 2:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt % N₂: 79 wt %-94 wt %

Saturated with H₂O

Test Suite 3:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt % N₂: 79 wt %-94 wt % CO₂: 5 wt %-30 wt % (2)Experimental Testing for Final Determination of Flammability Limits TestSuite 1:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt % N₂: 79 wt %-94 wt % Test Suite 2:

isoprene: 0 wt %-14 wt %

O₂: 6 wt %-21 wt % N₂: 79 wt %-94 wt %

Saturated with H₂O

Simulation software was used to give an estimate of the flammabilitycharacteristics of the system for several different testing conditions.CO₂ showed no significant affect on the system's flammability limits.Test suites 1 and 2 were confirmed by experimental testing. The modelingresults were in-line with the experimental test results. Only slightvariations were found with the addition of water.

The LOC was determined to be 9.5 vol % for an isoprene, O₂, N₂, and CO₂mixture at 40° C. and 1 atmosphere. The addition of up to 30% CO₂ didnot significantly affect the flammability characteristics of anisoprene, O₂, and N₂ mixture. Only slight variations in flammabilitycharacteristics were shown between a dry and water saturated isoprene,O₂, and N₂ system. The limiting temperature is about −54° C.Temperatures below about −54° C. are too low to propagate combustion ofisoprene.

In some embodiments, the LFL of isoprene ranges from about 1.5 vol. % toabout 2.0 vol %, and the UFL of isoprene ranges from about 2.0 vol. % toabout 12.0 vol. %, depending on the amount of oxygen in the system. Insome embodiments, the LOC is about 9.5 vol % oxygen. In someembodiments, the LFL of isoprene is between about 1.5 vol. % to about2.0 vol %, the UFL of isoprene is between about 2.0 vol. % to about 12.0vol. %, and the LOC is about 9.5 vol % oxygen when the temperature isbetween about 25° C. to about 55° C. (such as about 40° C.) and thepressure is between about 1 atmosphere and 3 atmospheres.

In some embodiments, isoprene is produced in the presence of less thanabout 9.5 vol % oxygen (that is, below the LOC required to have aflammable mixture of isoprene). In some embodiments in which isoprene isproduced in the presence of greater than or about 9.5 vol % oxygen, theisoprene concentration is below the LFL (such as below about 1.5 vol.%). For example, the amount of isoprene can be kept below the LFL bydiluting the isoprene composition with an inert gas (e.g., bycontinuously or periodically adding an inert gas such as nitrogen tokeep the isoprene composition below the LFL). In some embodiments inwhich isoprene is produced in the presence of greater than or about 9.5vol % oxygen, the isoprene concentration is above the UFL (such as aboveabout 12 vol. %). For example, the amount of isoprene can be kept abovethe UFL by using a system (such as any of the cell culture systemsdescribed herein) that produces isoprene at a concentration above theUFL. If desired, a relatively low level of oxygen can be used so thatthe UFL is also relatively low. In this case, a lower isopreneconcentration is needed to remain above the UFL.

In some embodiments in which isoprene is produced in the presence ofgreater than or about 9.5 vol % oxygen, the isoprene concentration iswithin the flammability envelope (such as between the LFL and the UFL).In some embodiments when the isoprene concentration may fall within theflammability envelope, one or more steps are performed to reduce theprobability of a fire or explosion. For example, one or more sources ofignition (such as any materials that may generate a spark) can beavoided. In some embodiments, one or more steps are performed to reducethe amount of time that the concentration of isoprene remains within theflammability envelope. In some embodiments, a sensor is used to detectwhen the concentration of isoprene is close to or within theflammability envelope. If desired, the concentration of isoprene can bemeasured at one or more time points during the culturing of cells, andthe cell culture conditions and/or the amount of inert gas can beadjusted using standard methods if the concentration of isoprene isclose to or within the flammability envelope. In particular embodiments,the cell culture conditions (such as fermentation conditions) areadjusted to either decrease the concentration of isoprene below the LFLor increase the concentration of isoprene above the UFL. In someembodiments, the amount of isoprene is kept below the LFL by dilutingthe isoprene composition with an inert gas (such as by continuously orperiodically adding an inert gas to keep the isoprene composition belowthe LFL).

In some embodiments, the amount of flammable volatiles other thanisoprene (such as one or more sugars) is at least about 2, 5, 10, 50,75, or 100-fold less than the amount of isoprene produced. In someembodiments, the portion of the gas phase other than isoprene gascomprises between about 0% to about 100% (volume) oxygen, such asbetween about 0% to about 10%, about 10% to about 20%, about 20% toabout 30%, about 30% to about 40%, about 40% to about 50%, about 50% toabout 60%, about 60% to about 70%, about 70% to about 80%, about 90% toabout 90%, or about 90% to about 100% (volume) oxygen. In someembodiments, the portion of the gas phase other than isoprene gascomprises between about 0% to about 99% (volume) nitrogen, such asbetween about 0% to about 10%, about 10% to about 20%, about 20% toabout 30%, about 30% to about 40%, about 40% to about 50%, about 50% toabout 60%, about 60% to about 70%, about 70% to about 80%, about 90% toabout 90%, or about 90% to about 99% (volume) nitrogen.

In some embodiments, the portion of the gas phase other than isoprenegas comprises between about 1% to about 50% (volume) CO₂, such asbetween about 1% to about 10%, about 10% to about 20%, about 20% toabout 30%, about 30% to about 40%, or about 40% to about 50% (volume)CO₂.

In some embodiments, an isoprene composition also contains ethanol. Forexample, ethanol may be used for extractive distillation of isoprene,resulting in compositions (such as intermediate product streams) thatinclude both ethanol and isoprene. Desirably, the amount of ethanol isoutside the flammability envelope for ethanol. The LOC of ethanol isabout 8.7 vol %, and the LFL for ethanol is about 3.3 vol % at standardconditions, such as about 1 atmosphere and about 60° F. (NFPA 69Standard on Explosion Prevention Systems, 2008 edition, which is herebyincorporated by reference in its entirety, particularly with respect toLOC, LFL, and UFL values). In some embodiments, compositions thatinclude isoprene and ethanol are produced in the presence of less thanthe LOC required to have a flammable mixture of ethanol (such as lessthan about 8.7% vol %). In some embodiments in which compositions thatinclude isoprene and ethanol are produced in the presence of greaterthan or about the LOC required to have a flammable mixture of ethanol,the ethanol concentration is below the LFL (such as less than about 3.3vol. %).

In various embodiments, the amount of oxidant (such as oxygen) is belowthe LOC of any fuel in the system (such as isoprene or ethanol). Invarious embodiments, the amount of oxidant (such as oxygen) is less thanabout 60, 40, 30, 20, 10, or 5% of the LOC of isoprene or ethanol. Invarious embodiments, the amount of oxidant (such as oxygen) is less thanthe LOC of isoprene or ethanol by at least 2, 4, 5, or more absolutepercentage points (vol %). In particular embodiments, the amount ofoxygen is at least 2 absolute percentage points (vol %) less than theLOC of isoprene or ethanol (such as an oxygen concentration of less than7.5 vol % when the LOC of isoprene is 9.5 vol %). In variousembodiments, the amount of fuel (such as isoprene or ethanol) is lessthan or about 25, 20, 15, 10, or 5% of the LFL for that fuel.

Exemplary Production of Isoprene

In some embodiments, the cells are cultured in a culture medium underconditions permitting the production of isoprene by the cells. By “peakabsolute productivity” is meant the maximum absolute amount of isoprenein the off-gas during the culturing of cells for a particular period oftime (e.g., the culturing of cells during a particular fermentationrun). By “peak absolute productivity time point” is meant the time pointduring a fermentation run when the absolute amount of isoprene in theoff-gas is at a maximum during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). In some embodiments, the isoprene amount is measuredat the peak absolute productivity time point. In some embodiments, thepeak absolute productivity for the cells is about any of the isopreneamounts disclosed herein.

By “peak specific productivity” is meant the maximum amount of isopreneproduced per cell during the culturing of cells for a particular periodof time (e.g., the culturing of cells during a particular fermentationrun). By “peak specific productivity time point” is meant the time pointduring the culturing of cells for a particular period of time (e.g., theculturing of cells during a particular fermentation run) when the amountof isoprene produced per cell is at a maximum. The specific productivityis determined by dividing the total productivity by the amount of cells,as determined by optical density at 600 nm (OD600). In some embodiments,the isoprene amount is measured at the peak specific productivity timepoint. In some embodiments, the peak specific productivity for the cellsis about any of the isoprene amounts per cell disclosed herein.

By “cumulative total productivity” is meant the cumulative, total amountof isoprene produced during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). In some embodiments, the cumulative, total amount ofisoprene is measured. In some embodiments, the cumulative totalproductivity for the cells is about any of the isoprene amountsdisclosed herein.

By “relative detector response” refers to the ratio between the detectorresponse (such as the GC/MS area) for one compound (such as isoprene) tothe detector response (such as the GC/MS area) of one or more compounds(such as all C5 hydrocarbons). The detector response may be measured asdescribed herein, such as the GC/MS analysis performed with an Agilent6890 GC/MS system fitted with an Agilent HP-5MS GC/MS column (30 m×250μm; 0.25 μm film thickness). If desired, the relative detector responsecan be converted to a weight percentage using the response factors foreach of the compounds. This response factor is a measure of how muchsignal is generated for a given amount of a particular compound (thatis, how sensitive the detector is to a particular compound). Thisresponse factor can be used as a correction factor to convert therelative detector response to a weight percentage when the detector hasdifferent sensitivities to the compounds being compared. Alternatively,the weight percentage can be approximated by assuming that the responsefactors are the same for the compounds being compared. Thus, the weightpercentage can be assumed to be approximately the same as the relativedetector response.

In some embodiments, the cells in culture produce isoprene at greaterthan or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600,700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000,5,000, or more nmole of isoprene/gram of cells for the wet weight of thecells/hour (nmole/g_(wcm)/hr). In some embodiments, the amount ofisoprene is between about 2 to about 5,000 nmole/g_(wcm)/hr, such asbetween about 2 to about 100 nmole/g_(wcm)/hr, about 100 to about 500nmole/g_(wcm)/hr, about 150 to about 500 nmole/g_(wcm)/hr, about 500 toabout 1,000 nmole/g_(wcm)/hr, about 1,000 to about 2,000nmole/g_(wcm)/hr, or about 2,000 to about 5,000 nmole/g_(wcm)/hr. Insome embodiments, the amount of isoprene is between about 20 to about5,000 nmole/g_(wcm)/hr, about 100 to about 5,000 nmole/g_(wcm)/hr, about200 to about 2,000 nmole/g_(wcm)/hr, about 200 to about 1,000nmole/g_(wcm)/hr, about 300 to about 1,000 nmole/g_(wcm)/hr, or about400 to about 1,000 nmole/g_(wcm)/hr.

The amount of isoprene in units of nmole/g_(wcm)/hr can be measured asdisclosed in U.S. Pat. No. 5,849,970, which is hereby incorporated byreference in its entirety, particularly with respect to the measurementof isoprene production. For example, two mL of headspace (e.g.,headspace from a culture such as 2 mL of culture cultured in sealedvials at 320 C with shaking at 200 rpm for approximately 3 hours) areanalyzed for isoprene using a standard gas chromatography system, suchas a system operated isothermally (850 C) with an n-octane/porasil Ccolumn (Alltech Associates, Inc., Deerfield, Ill.) and coupled to a RGD2mercuric oxide reduction gas detector (Trace Analytical, Menlo Park,Calif.) (see, for example, Greenberg et al, Atmos. Environ. 27A:2689-2692, 1993; Silver et al., Plant Physiol. 97:1588-1591, 1991, whichare each hereby incorporated by reference in their entireties,particularly with respect to the measurement of isoprene production).The gas chromatography area units are converted to nmol isoprene via astandard isoprene concentration calibration curve. In some embodiments,the value for the grams of cells for the wet weight of the cells iscalculated by obtaining the A₆₀₀ value for a sample of the cell culture,and then converting the A₆₀₀ value to grams of cells based on acalibration curve of wet weights for cell cultures with a known A₆₀₀value. In some embodiments, the grams of the cells is estimated byassuming that one liter of broth (including cell medium and cells) withan A₆₀₀ value of 1 has a wet cell weight of 1 gram. The value is alsodivided by the number of hours the culture has been incubating for, suchas three hours.

In some embodiments, the cells in culture produce isoprene at greaterthan or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600,700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000,5,000, 10,000, 100,000, or more ng of isoprene/gram of cells for the wetweight of the cells/hr (ng/g_(wcm)/h). In some embodiments, the amountof isoprene is between about 2 to about 5,000 ng/g_(wcm)/h, such asbetween about 2 to about 100 ng/g_(wcm)/h, about 100 to about 500ng/g_(wcm)/h, about 500 to about 1,000 ng/g_(wcm)/h, about 1,000 toabout 2,000 ng/g_(wcm)/h, or about 2,000 to about 5,000 ng/g_(wcm)/h. Insome embodiments, the amount of isoprene is between about 20 to about5,000 ng/g_(wcm)/h, about 100 to about 5,000 ng/g_(wcm)/h, about 200 toabout 2,000 ng/g_(wcm)/h, about 200 to about 1,000 ng/g_(wcm)/h, about300 to about 1,000 ng/g_(wcm)/h, or about 400 to about 1,000ng/g_(wcm)/h. The amount of isoprene in ng/g_(wcm)/h can be calculatedby multiplying the value for isoprene production in the units ofnmole/g_(wcm)/hr discussed above by 68.1 (as described in Equation 5below).

In some embodiments, the cells in culture produce a cumulative titer(total amount) of isoprene at greater than or about 1, 10, 25, 50, 100,150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500,1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, ormore mg of isoprene/L of broth (mg/L_(broth), wherein the volume ofbroth includes the volume of the cells and the cell medium). In someembodiments, the amount of isoprene is between about 2 to about 5,000mg/L_(broth), such as between about 2 to about 100 mg/L_(broth), about100 to about 500 mg/L_(broth), about 500 to about 1,000 mg/L_(broth),about 1,000 to about 2,000 mg/L_(broth), or about 2,000 to about 5,000mg/L_(broth). In some embodiments, the amount of isoprene is betweenabout 20 to about 5,000 mg/L_(broth), about 100 to about 5,000mg/L_(broth), about 200 to about 2,000 mg/L_(broth), about 200 to about1,000 mg/L_(broth), about 300 to about 1,000 mg/L_(broth), or about 400to about 1,000 mg/L_(broth).

The specific productivity of isoprene in mg of isoprene/L of headspacefrom shake flask or similar cultures can be measured by taking a 1 mlsample from the cell culture at an OD₆₀₀ value of approximately 1.0,putting it in a 20 mL vial, incubating for 30 minutes, and thenmeasuring the amount of isoprene in the headspace (as described, forexample, in Example I, part II). If the OD₆₀₀ value is not 1.0, then themeasurement can be normalized to an OD₆₀₀ value of 1.0 by dividing bythe OD₆₀₀ value. The value of mg isoprene/L headspace can be convertedto mg/L_(broth)/hr/OD₆₀₀ of culture broth by multiplying by a factor of38. The value in units of mg/L_(broth)/hr/OD₆₀₀ can be multiplied by thenumber of hours and the OD₆₀₀ value to obtain the cumulative titer inunits of mg of isoprene/L of broth.

The instantaneous isoprene production rate in mg/L_(broth)/hr in afermentor can be measured by taking a sample of the fermentor off-gas,analyzing it for the amount of isoprene (in units such as mg of isopreneper L_(gas)) as described, for example, in Example I, part II andmultiplying this value by the rate at which off-gas is passed thougheach liter of broth (e.g., at 1 vvm (volume of air/volume ofbroth/minute) this is 60 L_(gas) per hour). Thus, an off-gas level of 1mg/L_(gas) corresponds to an instantaneous production rate of 60mg/L_(broth)/hr at air flow of 1 vvm. If desired, the value in the unitsmg/L_(broth)/hr can be divided by the OD₆₀₀ value to obtain the specificrate in units of mg/L_(broth)/hr/OD. The average value of mgisoprene/L_(gas) can be converted to the total product productivity(grams of isoprene per liter of fermentation broth, mg/L_(broth)) bymultiplying this average off-gas isoprene concentration by the totalamount of off-gas sparged per liter of fermentation broth during thefermentation. Thus, an average off-gas isoprene concentration of 0.5mg/L_(broth)/hr over 10 hours at 1 vvm corresponds to a total productconcentration of 300 mg isoprene/L_(broth).

In some embodiments, the cells in culture convert greater than or about0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0,3.5, 4.0, 5.0, 6.0, 7.0, or 8.0% of the carbon in the cell culturemedium into isoprene. In some embodiments, the percent conversion ofcarbon into isoprene is between such as about 0.002 to about 4.0%, about0.002 to about 3.0%, about 0.002 to about 2.0%, about 0.002 to about1.6%, about 0.002 to about 0.005%, about 0.005 to about 0.01%, about0.01 to about 0.05%, about 0.05 to about 0.15%, 0.15 to about 0.2%,about 0.2 to about 0.3%, about 0.3 to about 0.5%, about 0.5 to about0.8%, about 0.8 to about 1.0%, or about 1.0 to about 1.6%. In someembodiments, the percent conversion of carbon into isoprene is betweenabout 0.002 to about 0.4%, 0.002 to about 0.16%, 0.04 to about 0.16%,about 0.005 to about 0.3%, about 0.01 to about 0.3%, or about 0.05 toabout 0.3%.

The percent conversion of carbon into isoprene (also referred to as “%carbon yield”) can be measured by dividing the moles carbon in theisoprene produced by the moles carbon in the carbon source (such as themoles of carbon in batched and fed glucose and yeast extract). Thisnumber is multiplied by 100% to give a percentage value (as indicated inEquation 1).

% Carbon Yield=(moles carbon in isoprene produced)/(moles carbon incarbon source)*100  Equation 1

For this calculation, yeast extract can be assumed to contain 50% w/wcarbon. As an example, for the 500 liter described in Example 7, partVIII, the percent conversion of carbon into isoprene can be calculatedas shown in Equation 2.

% Carbon Yield=(39.1 g isoprene*1/68.1 mol/g*5 C/mol)/[(181221 gglucose*1/180 mol/g*6 C/mol)+(17780 g yeast extract*0.5*1/12mol/g)]*100=0.042%  Equation 2

For the two 500 liter fermentations described herein (Example 7, partsVII and VIII), the percent conversion of carbon into isoprene wasbetween 0.04-0.06%. A 0.11-0.16% carbon yield has been achieved using 14liter systems as described herein. Example 11, part V describes the1.53% conversion of carbon to isoprene using the methods describedherein.

One skilled in the art can readily convert the rates of isopreneproduction or amount of isoprene produced into any other units.Exemplary equations are listed below for interconverting between units.

Units for Rate of Isoprene production (total and specific)

1 g isoprene/L_(broth)/hr=14.7 mmol isoprene/L_(broth)/hr (totalvolumetric rate)  Equation 3

1 nmol isoprene/g_(wcm)/hr=1 nmol isoprene/L_(broth)/hr/OD₆₀₀ (Thisconversion assumes that one liter of broth with an OD₆₀₀ value of 1 hasa wet cell weight of 1 gram.)  Equation 4

1 nmol isoprene/g_(wcm)/hr=68.1 ng isoprene/g_(wcm)/hr (given themolecular weight of isoprene)  Equation 5

1 nmol isoprene/L_(gas) O₂/hr=90 nmol isoprene/L_(broth)/hr (at an O₂flow rate of 90 L/hr per L of culture broth)  Equation 6

1 ug isoprene/L_(gas) isoprene in off-gas=60 ug isoprene/L_(broth)/hr ata flow rate of 60 L_(gas) per L_(broth) (1 vvm)  Equation 7

Units for Titer (Total and Specific)

1 nmol isoprene/mg cell protein=150 nmol isoprene/L_(broth)/OD₆₀₀ (Thisconversion assumes that one liter of broth with an OD₆₀₀ value of 1 hasa total cell protein of approximately 150 mg) (specificproductivity)  Equation 8

1 g isoprene/L_(broth)=14.7 mmol isoprene/L_(broth) (totaltiter)  Equation 9

If desired, Equation 10 can be used to convert any of the units thatinclude the wet weight of the cells into the corresponding units thatinclude the dry weight of the cells.

Dry weight of cells=(wet weight of cells)/3.3  Equation 10

If desired, Equation 11 can be used to convert between units of ppm andug/L. In particular, “ppm” means parts per million defined in terms ofug/g (w/w) or uL/L (vol/vol). Conversion of ug/L to ppm (e.g., ug ofanalyte per g of gas) can be performed by determining the mass per L ofoff-gas (i.e., the density of the gas). For example, a liter of air atSTP has a density of approximately 1.2 g/L. Thus, a concentration of 1ppm (ug/g) equals 0.83 ug/L at STP (equation 11). The conversion of ppm(ug/g) to ug/L is a function of both pressure, temperature, and overallcomposition of the off-gas.

1 ppm (ug/g) equals 0.83 ug/L at standard temperature and pressure (STP;101.3 kPa (1 bar) and 273.15K).  Equation 11

Conversion of ug/L to ppmv (e.g., uL of analyte per L of gas) can beperformed using the Universal Gas Law (equation 12). For example, anoff-gas concentration of 1000 ug/L_(gas) corresponds to 14.7umol/L_(gas). The universal gas constant is 0.082057 L·atm K⁻¹mol⁻¹, sousing equation 12, the volume occupied by 14.7 umol of HG at STP isequal to 0.329 mL. Therefore, the concentration of 1000 ug/L HG is equalto 329 ppmv or 0.0329% (v/v) at STP.

PV=nRT, where “P” is pressure, “V” is volume, “n” is moles of gas, “R”is the Universal gas constant, and “T” is temperature inKelvin.  Equation 12

The amount of impurities in isoprene compositions are typically measuredherein on a weight per volume (w/v) basis in units such as ug/L. Ifdesired, measurements in units of ug/L can be converted to units ofmg/m³ using equation 13.

1 ug/L=1 mg/m³  Equation 13

In some embodiments encompassed by the invention, a cell comprising aheterologous nucleic acid encoding an isoprene synthase polypeptideproduces an amount of isoprene that is at least or about 2-fold, 3-fold,5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold,400-fold, or greater than the amount of isoprene produced from acorresponding cell grown under essentially the same conditions withoutthe heterologous nucleic acid encoding the isoprene synthasepolypeptide.

In some embodiments encompassed by the invention, a cell comprising aheterologous nucleic acid encoding an isoprene synthase polypeptide andone or more heterologous nucleic acids encoding a DXS, IDI, and/or MVApathway polypeptide produces an amount of isoprene that is at least orabout 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold,150-fold, 200-fold, 400-fold, or greater than the amount of isopreneproduced from a corresponding cell grown under essentially the sameconditions without the heterologous nucleic acids.

In some embodiments, the isoprene composition comprises greater than orabout 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weightcompared to the total weight of all C5 hydrocarbons in the composition.In some embodiments, the composition has a relative detector response ofgreater than or about 99.90, 99.91, 99.92, 99.93, 99.94, 99.95, 99.96,99.97, 99.98, 99.99, or 100% for isoprene compared to the detectorresponse for all C5 hydrocarbons in the composition. In someembodiments, the isoprene composition comprises between about 99.90 toabout 99.92, about 99.92 to about 99.94, about 99.94 to about 99.96,about 99.96 to about 99.98, about 99.98 to 100% isoprene by weightcompared to the total weight of all C5 hydrocarbons in the composition.

In some embodiments, the isoprene composition comprises less than orabout 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005,0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,trans-piperylene, cis-piperylene, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared tothe total weight of all C5 hydrocarbons in the composition. In someembodiments, the composition has a relative detector response of lessthan or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001,0.0005, 0.0001, 0.00005, or 0.00001% for C5 hydrocarbons other thanisoprene compared to the detector response for all C5 hydrocarbons inthe composition. In some embodiments, the composition has a relativedetector response of less than or about 0.12, 0.10, 0.08, 0.06, 0.04,0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,trans-piperylene, cis-piperylene, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne compared to the detectorresponse for all C5 hydrocarbons in the composition. In someembodiments, the isoprene composition comprises between about 0.02 toabout 0.04%, about 0.04 to about 0.06%, about 0.06 to 0.08%, about 0.08to 0.10%, or about 0.10 to about 0.12% C5 hydrocarbons other thanisoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene,trans-1,3-pentadiene, 1-pentyne, 2-pentyne, 1-pentene,2-methyl-1-butene, 3-methyl-1-butyne, trans-piperylene, cis-piperylene,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) byweight compared to the total weight of all C5 hydrocarbons in thecomposition.

In some embodiments, the isoprene composition comprises less than orabout 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ug/L of acompound that inhibits the polymerization of isoprene for any compoundin the composition that inhibits the polymerization of isoprene. In someembodiments, the isoprene composition comprises between about 0.005 toabout 50, such as about 0.01 to about 10, about 0.01 to about 5, about0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about 0.005ug/L of a compound that inhibits the polymerization of isoprene for anycompound in the composition that inhibits the polymerization ofisoprene. In some embodiments, the isoprene composition comprises lessthan or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005ug/L of a hydrocarbon other than isoprene (such 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1-pentyne, 2-pentyne,1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, trans-piperylene,cis-piperylene, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne). In some embodiments, the isoprene compositioncomprises between about 0.005 to about 50, such as about 0.01 to about10, about 0.01 to about 5, about 0.01 to about 1, about 0.01 to about0.5, or about 0.01 to about 0.005 ug/L of a hydrocarbon other thanisoprene. In some embodiments, the isoprene composition comprises lessthan or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005ug/L of a protein or fatty acid (such as a protein or fatty acid that isnaturally associated with natural rubber).

In some embodiments, the isoprene composition comprises less than orabout 10, 5, 1, 0.8, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of alphaacetylenes, piperylenes, acetonitrile, or 1,3-cyclopentadiene. In someembodiments, the isoprene composition comprises less than or about 5, 1,0.5, 0.1, 0.05, 0.01, or 0.005 ppm of sulfur or allenes. In someembodiments, the isoprene composition comprises less than or about 30,20, 15, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of all acetylenes(such as pentyne-1, butyne-2, 2MB1-3yne, and 1-pentyne-4-yne). In someembodiments, the isoprene composition comprises less than or about 2000,1000, 500, 200, 100, 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or0.005 ppm of isoprene dimers, such as cyclic isoprene dimmers (e.g.,cyclic C10 compounds derived from the dimerization of two isopreneunits).

In some embodiments, the isoprene composition includes ethanol, acetone,a C5 prenyl alcohol (such as 3-methyl-3-buten-1-ol or3-methyl-2-buten-1-ol), or any two or more of the foregoing. Inparticular embodiments, the isoprene composition comprises greater thanor about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100,or 120 ug/L of ethanol, acetone, a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol), or any two or more ofthe foregoing. In some embodiments, the isoprene composition comprisesbetween about 0.005 to about 120, such as about 0.01 to about 80, about0.01 to about 60, about 0.01 to about 40, about 0.01 to about 30, about0.01 to about 20, about 0.01 to about 10, about 0.1 to about 80, about0.1 to about 60, about 0.1 to about 40, about 5 to about 80, about 5 toabout 60, or about 5 to about 40 ug/L of ethanol, acetone, a C5 prenylalcohol, or any two or more of the foregoing.

In some embodiments, the isoprene composition includes one or more ofthe following components: 2-heptanone, 6-methyl-5-hepten-2-one,2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal,acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl,2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol,3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone,3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butylacetate, 3-methylbutyl acetate, 3-methyl-3-but-1-enyl acetate,3-methyl-2-but-1-enyl acetate, (E)-3,7-dimethyl-1,3,6-octatriene,(Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or alinear isoprene polymer (such as a linear isoprene dimer or a linearisoprene trimer derived from the polymerization of multiple isopreneunits). In various embodiments, the amount of one of these componentsrelative to amount of isoprene in units of percentage by weight (i.e.,weight of the component divided by the weight of isoprene times 100) isgreater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, or 110% (w/w). In some embodiments, therelative detector response for the second compound compared to thedetector response for isoprene is greater than or about 0.01, 0.02,0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110%.In various embodiments, the amount of one of these components relativeto amount of isoprene in units of percentage by weight (i.e., weight ofthe component divided by the weight of isoprene times 100) is betweenabout 0.01 to about 105% (w/w), such as about 0.01 to about 90, about0.01 to about 80, about 0.01 to about 50, about 0.01 to about 20, about0.01 to about 10, about 0.02 to about 50, about 0.05 to about 50, about0.1 to about 50, or 0.1 to about 20% (w/w).

In some embodiments, the isoprene composition includes one or more ofthe following: an alcohol, an aldehyde, or a ketone (such as any of thealcohols, aldehyes, or ketones described herein). In some embodiments,the isoprene composition includes (i) an alcohol and an aldehyde, (ii)an alcohol and a ketone, (iii) an aldehyde and a ketone, or (iv) analcohol, an aldehyde, and a ketone.

In some embodiments, the isoprene composition contains one or more ofthe following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol,3-methyl-1-propanol, acetone, acetic acid, 2-butanone,2-methyl-1-butanol, or indole. In some embodiments, the isoprenecomposition contains 1 ppm or more of one or more of the following:methanol, acetaldehyde, ethanol, methanethiol, 1-butanol,3-methyl-1-propanol, acetone, acetic acid, 2-butanone,2-methyl-1-butanol, or indole. In some embodiments, the concentration ofmore of one or more of the following: methanol, acetaldehyde, ethanol,methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid,2-butanone, 2-methyl-1-butanol, or indole, is between about 1 to about10,000 ppm in an isoprene composition (such as off-gas before it ispurified). In some embodiments, the isoprene composition (such asoff-gas after it has undergone one or more purification steps) includesone or more of the following: methanol, acetaldehyde, ethanol,methanethiol, 1-butanol, 3-methyl-1-propanol, acetone, acetic acid,2-butanone, 2-methyl-1-butanol, or indole, at a concentration betweenabout 1 to about 100 ppm, such as about 1 to about 10 ppm, about 10 toabout 20 ppm, about 20 to about 30 ppm, about 30 to about 40 ppm, about40 to about 50 ppm, about 50 to about 60 ppm, about 60 to about 70 ppm,about 70 to about 80 ppm, about 80 to about 90 ppm, or about 90 to about100 ppm. Volatile organic compounds from cell cultures (such as volatileorganic compounds in the headspace of cell cultures) can be analyzedusing standard methods such as those described herein or other standardmethods such as proton transfer reaction-mass spectrometry (see, forexample, Bunge et al., Applied and Environmental Microbiology,74(7):2179-2186, 2008 which is hereby incorporated by reference in itsentirety, particular with respect to the analysis of volatile organiccompounds).

In some embodiments, the composition comprises greater than about 2 mgof isoprene, such as greater than or about 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg ofisoprene. In some embodiments, the composition comprises greater than orabout 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene. Insome embodiments, the amount of isoprene in the composition is betweenabout 2 to about 5,000 mg, such as between about 2 to about 100 mg,about 100 to about 500 mg, about 500 to about 1,000 mg, about 1,000 toabout 2,000 mg, or about 2,000 to about 5,000 mg. In some embodiments,the amount of isoprene in the composition is between about 20 to about5,000 mg, about 100 to about 5,000 mg, about 200 to about 2,000 mg,about 200 to about 1,000 mg, about 300 to about 1,000 mg, or about 400to about 1,000 mg. In some embodiments, greater than or about 20, 25,30, 40, 50, 60, 70, 80, 90, or 95% by weight of the volatile organicfraction of the composition is isoprene.

In some embodiments, the composition includes ethanol. In someembodiments, the composition includes between about 75 to about 90% byweight of ethanol, such as between about 75 to about 80%, about 80 toabout 85%, or about 85 to about 90% by weight of ethanol. In someembodiments in which the composition includes ethanol, the compositionalso includes between about 4 to about 15% by weight of isoprene, suchas between about 4 to about 8%, about 8 to about 12%, or about 12 toabout 15% by weight of isoprene.

In some embodiments encompassed by the invention, a cell comprising oneor more heterologous nucleic acids encoding an isoprene synthasepolypeptide, DXS polypeptide, IDI polypeptide, and/or MVA pathwaypolypeptide produces an amount of an isoprenoid compound (such as acompound with 10 or more carbon atoms that is formed from the reactionof one or more IPP molecules with one or more DMAPP molecules) that isgreater than or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold,100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount ofthe isoprenoid compound produced from a corresponding cell grown underessentially the same conditions without the one or more heterologousnucleic acids. In some embodiments encompassed by the invention, a cellcomprising one or more heterologous nucleic acids encoding an isoprenesynthase polypeptide, DXS polypeptide, IDI polypeptide, and/or MVApathway polypeptide produces an amount of a C5 prenyl alcohol (such as3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol) that is greater than orabout 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold,150-fold, 200-fold, 400-fold, or greater than the amount of the C5prenyl alcohol produced from a corresponding cell grown underessentially the same conditions without the one or more heterologousnucleic acids.

Exemplary Isoprene Purification Methods

In some embodiments, any of the methods described herein further includerecovering the isoprene. For example, the isoprene produced using thecompositions and methods of the invention can be recovered usingstandard techniques. such as gas stripping, membrane enhancedseparation, fractionation, adsorption/desorption, pervaporation, thermalor vacuum desorption of isoprene from a solid phase, or extraction ofisoprene immobilized or absorbed to a solid phase with a solvent (see,for example, U.S. Pat. Nos. 4,703,007 and 4,570,029, which are eachhereby incorporated by reference in their entireties, particularly withrespect to isoprene recovery and purification methods). In particular,embodiments, extractive distillation with an alcohol (such as ethanol,methanol, propanol, or a combination thereof) is used to recover theisoprene. In some embodiments, the recovery of isoprene involves theisolation of isoprene in a liquid form (such as a neat solution ofisoprene or a solution of isoprene in a solvent). Gas stripping involvesthe removal of isoprene vapor from the fermentation off-gas stream in acontinuous manner. Such removal can be achieved in several differentways including, but not limited to, adsorption to a solid phase,partition into a liquid phase, or direct condensation (such ascondensation due to exposure to a condensation coil or do to an increasein pressure). In some embodiments, membrane enrichment of a diluteisoprene vapor stream above the dew point of the vapor resulting in thecondensation of liquid isoprene. In some embodiments, the isoprene iscompressed and condensed.

The recovery of isoprene may involve one step or multiple steps. In someembodiments, the removal of isoprene vapor from the fermentation off-gasand the conversion of isoprene to a liquid phase are performedsimultaneously. For example, isoprene can be directly condensed from theoff-gas stream to form a liquid. In some embodiments, the removal ofisoprene vapor from the fermentation off-gas and the conversion ofisoprene to a liquid phase are performed sequentially. For example,isoprene may be adsorbed to a solid phase and then extracted from thesolid phase with a solvent.

In some embodiments, any of the methods described herein further includepurifying the isoprene. For example, the isoprene produced using thecompositions and methods of the invention can be purified using standardtechniques. Purification refers to a process through which isoprene isseparated from one or more components that are present when the isopreneis produced. In some embodiments, the isoprene is obtained as asubstantially pure liquid. Examples of purification methods include (i)distillation from a solution in a liquid extractant and (ii)chromatography. As used herein, “purified isoprene” means isoprene thathas been separated from one or more components that are present when theisoprene is produced. In some embodiments, the isoprene is at leastabout 20%, by weight, free from other components that are present whenthe isoprene is produced. In various embodiments, the isoprene is atleast or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%,by weight, pure. Purity can be assayed by any appropriate method, e.g.,by column chromatography, HPLC analysis, or GC-MS analysis.

In some embodiments, at least a portion of the gas phase remaining afterone or more recovery steps for the removal of isoprene is recycled byintroducing the gas phase into a cell culture system (such as afermentor) for the production of isoprene.

In some embodiments, any of the methods described herein further includepolymerizing the isoprene. For example, standard methods can be used topolymerize the purified isoprene to form cis-polyisoprene or other downstream products using standard methods. Accordingly, the invention alsofeatures a tire comprising polyisoprene, such as cis-1,4-polyisopreneand/or trans-1,4-polyisoprene made from any of the isoprene compositionsdisclosed herein.

EXAMPLES

The examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above. Unless indicated otherwise, temperature is in degreesCentigrade and pressure is at or near atmospheric. The foregoingexamples and detailed description are offered by way of illustration andnot by way of limitation. All publications, patent applications, andpatents cited in this specification are herein incorporated by referenceas if each individual publication, patent application, or patent werespecifically and individually indicated to be incorporated by reference.In particular, all publications cited herein are expressly incorporatedherein by reference for the purpose of describing and disclosingcompositions and methodologies which might be used in connection withthe invention. Although the foregoing invention has been described insome detail by way of illustration and example for purposes of clarityof understanding, it will be readily apparent to those of ordinary skillin the art in light of the teachings of this invention that certainchanges and modifications may be made thereto without departing from thespirit or scope of the appended claims.

Example 1 Production of Isoprene in E. Coli Expressing Recombinant KudzuIsoprene Synthase I. Construction of Vectors for Expression of the KudzuIsoprene Synthase in E. Coli

The protein sequence for the kudzu (Pueraria montana) isoprene synthasegene (IspS) was obtained from GenBank (AAQ84170). A kudzu isoprenesynthase gene, optimized for E. coli codon usage, was purchased fromDNA2.0 (SEQ ID NO:1). The isoprene synthase gene was removed from thesupplied plasmid by restriction endonuclease digestion withBspLU11I/PstI, gel-purified, and ligated into pTrcHis2B (Invitrogen)that had been digested with NcoI/PstI. The construct was designed suchthat the stop codon in the isoprene synthase gene 5′ to the PstI site.As a result, when the construct was expressed the His-Tag is notattached to the isoprene synthase protein. The resulting plasmid,pTrcKudzu, was verified by sequencing (FIGS. 2 and 3).

The isoprene synthase gene was also cloned into pET16b (Novagen). Inthis case, the isoprene synthase gene was inserted into pET16b such thatthe recombinant isoprene synthase protein contained the N-terminal Histag. The isoprene synthase gene was amplified from pTrcKudzu by PCRusing the primer set pET-His-Kudzu-2F:5′-CGTGAGATCATATGTGTGCGACCTCTTCTCAATTTAC (SEQ ID NO:3) andpET-His-Kudzu-R: 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ IDNO:4). These primers added an NdeI site at the 5′-end and a BamH1 siteat the 3′ end of the gene respectively. The plasmid pTrcKudzu, describedabove, was used as template DNA, Herculase polymerase (Stratagene) wasused according to manufacture's directions, and primers were added at aconcentration of 10 pMols. The PCR was carried out in a total volume of25 μl. The PCR product was digested with NdeI/BamH1 and cloned intopET16b digested with the same enzymes. The ligation mix was transformedinto E. coli Top10 (Invitrogen) and the correct clone selected bysequencing. The resulting plasmid, in which the kudzu isoprene synthasegene was expressed from the T7 promoter, was designated pETNHisKudzu(FIGS. 4 and 5).

The kudzu isoprene synthase gene was also cloned into the low copynumber plasmid pCL1920. Primers were used to amplify the kudzu isoprenesynthase gene from pTrcKudzu described above. The forward primer added aHindIII site and an E. coli consensus RBS to the 5′ end. The PstIcloning site was already present in pTrcKudzu just 3′ of the stop codonso the reverse primer was constructed such that the final PCR productincludes the PstI site. The sequences of the primers were:HindIII-rbs-Kudzu F: 5′-CATATGAAAGCTTGTATCGATTAAATAAGGAGGAATAAACC (SEQID NO:6) and BamH1-Kudzu R:

5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:4). The PCR productwas amplified using Herculase polymerase with primers at a concentrationof 10 μmol and with 1 ng of template DNA (pTrcKudzu). The amplificationprotocol included 30 cycles of (95° C. for 1 minute, 60° C. for 1minute, 72° C. for 2 minutes). The product was digested with HindIII andPstI and ligated into pCL1920 which had also been digested with HindIIIand PstI. The ligation mix was transformed into E. coli Top10. Severaltransformants were checked by sequencing. The resulting plasmid wasdesignated pCL-lac-Kudzu (FIGS. 6 and 7).

II. Determination of Isoprene Production

For the shake flask cultures, one ml of a culture was transferred fromshake flasks to 20 ml CTC headspace vials (Agilent vial cat#5188 2753;cap cat#5188 2759). The cap was screwed on tightly and the vialsincubated at the equivalent temperature with shaking at 250 rpm. After30 minutes the vials were removed from the incubator and analyzed asdescribed below (see Table 1 for some experimental values from thisassay).

In cases where isoprene production in fermentors was determined, sampleswere taken from the off-gas of the fermentor and analyzed directly asdescribed below (see Table 2 for some experimental values from thisassay).

The analysis was performed using an Agilent 6890 GC/MS system interfacedwith a CTC Analytics (Switzerland) CombiPAL autosampler operating inheadspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μmfilm thickness) was used for separation of analytes. The sampler was setup to inject 500 μL of headspace gas. The GC/MS method utilized heliumas the carrier gas at a flow of 1 ml/min. The injection port was held at250° C. with a split ratio of 50:1. The oven temperature was held at 37°C. for the 2 minute duration of the analysis. The Agilent 5793N massselective detector was run in single ion monitoring (SIM) mode on m/z67. The detector was switched off from 1.4 to 1.7 minutes to allow theelution of permanent gases. Under these conditions isoprene(2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. Acalibration table was used to quantify the absolute amount of isopreneand was found to be linear from 1 μg/L to 2000 μg/L. The limit ofdetection was estimated to be 50 to 100 ng/L using this method.

III. Production of Isoprene in Shake Flasks Containing E. Coli CellsExpressing Recombinant Isoprene Synthase

The vectors described above were introduced to E. coli strain BL21(Novagen) to produce strains BL21/ptrcKudzu, BL21/pCL-lac-Kudzu andBL21/pETHisKudzu. The strains were spread for isolation onto LA (Luriaagar)+carbenicillin (50 μg/ml) and incubated overnight at 37° C. Singlecolonies were inoculated into 250 ml baffled shake flasks containing 20ml Luria Bertani broth (LB) and carbenicillin (100 μg/ml). Cultures weregrown overnight at 20° C. with shaking at 200 rpm. The OD₆₀₀ of theovernight cultures were measured and the cultures were diluted into a250 ml baffled shake flask containing 30 ml MagicMedia(Invitrogen)+carbenicillin (100 μg/ml) to an OD₆₀₀˜0.05. The culture wasincubated at 30° C. with shaking at 200 rpm. When the OD₆₀₀˜0.5-0.8, 400μM IPTG was added and the cells were incubated for a further 6 hours at30° C. with shaking at 200 rpm. At 0, 2, 4 and 6 hours after inductionwith IPTG, 1 ml aliquots of the cultures were collected, the OD₆₀₀ wasdetermined and the amount of isoprene produced was measured as describedabove. Results are shown in FIG. 8.

IV. Production of Isoprene from BL21/ptrcKudzu in 14 Liter Fermentation

Large scale production of isoprene from E. coli containing therecombinant kudzu isoprene synthase gene was determined from a fed-batchculture. The recipe for the fermentation media (TM2) per liter offermentation medium was as follows: K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g,Mg504*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3g, (NH₄)₂SO₄ 3.2 g, yeast extract 5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. The pH was adjusted to 6.8 with potassium hydroxide (KOH) andq.s. to volume. The final product was filter sterilized with 0.22μfilter (only, do not autoclave). The recipe for 1000× Modified TraceMetal Solution was as follows: Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g,NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄ 5H₂O 100mg, H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. Each component was dissolved oneat a time in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume andfilter sterilized with a 0.22μ filter.

This experiment was carried out in 14 L bioreactor to monitor isopreneformation from glucose at the desired fermentation, pH 6.7 andtemperature 34° C. An inoculum of E. coli strain BL21/ptrcKudzu takenfrom a frozen vial was prepared in soytone-yeast extract-glucose medium.After the inoculum grew to OD₅₅₀=0.6, two 600 ml flasks were centrifugedand the contents resuspended in 70 ml supernatant to transfer the cellpellet (70 ml of OD 3.1 material) to the bioreactor. At various timesafter inoculation, samples were removed and the amount of isopreneproduced was determined as described above. Results are shown in FIG. 9.

Example 2 Production of Isoprene in E. Coli Expressing RecombinantPoplar Isoprene Synthase

The protein sequence for the poplar (Populus alba×Populus tremula)isoprene synthase (Schnitzler, J-P, et al. (2005) Planta 222:777-786)was obtained from GenBank (CAC35696). A gene, codon optimized for E.coli, was purchased from DNA2.0 (p9796-poplar, FIGS. 30 and 31). Theisoprene synthase gene was removed from the supplied plasmid byrestriction endonuclease digestion with BspLU11I/PstI, gel-purified, andligated into pTrcHis2B that had been digested with NcoI/PstI. Theconstruct is cloned such that the stop codon in the insert is before thePstI site, which results in a construct in which the His-Tag is notattached to the isoprene synthase protein. The resulting plasmidpTrcPoplar (FIGS. 32 and 33), was verified by sequencing.

Example 3 Production of Isoprene in Panteoa citrea ExpressingRecombinant Kudzu Isoprene Synthase

The pTrcKudzu and pCL-lac Kudzu plasmids described in Example 1 wereelectroporated into P. citrea (U.S. Pat. No. 7,241,587). Transformantswere selected on LA containing carbenicillin (200 μg/ml) orspectinomycin (50 μg/ml) respectively. Production of isoprene from shakeflasks and determination of the amount of isoprene produced wasperformed as described in Example 1 for E. coli strains expressingrecombinant kudzu isoprene synthase. Results are shown in FIG. 10.

Example 4 Production of Isoprene in Bacillus subtilis ExpressingRecombinant Kudzu Isoprene Synthase

I. Construction of a B. subtilis replicating plasmid for the expressionof kudzu isoprene synthase

The kudzu isoprene synthase gene was expressed in Bacillus subtilisaprEnprE Pxyl-comK strain (BG3594comK) using a replicating plasmid(pBS19 with a chloramphenicol resistance cassette) under control of theaprE promoter. The isoprene synthase gene, the aprE promoter and thetranscription terminator were amplified separately and fused using PCR.The construct was then cloned into pBS 19 and transformed into B.subtilis.

a) Amplification of the aprE Promoter

The aprE promoter was amplified from chromosomal DNA from Bacillussubtilis using the following primers:

CF 797 (+) Start aprE promoter MfeI (SEQ ID NO: 58)5′-GACATCAATTGCTCCATTTTCTTCTGCTATCCF 07-43 (−) Fuse aprE promoter to Kudzu ispS (SEQ ID NO: 59)5′-ATTGAGAAGAGGTCGCACACACTCTTTACCCTCTCCTTTTA

b) Amplification of the Isoprene Synthase Gene

The kudzu isoprene synthase gene was amplified from plasmid pTrcKudzu(SEQ ID NO:2). The gene had been codon optimized for E. coli andsynthesized by DNA 2.0. The following primers were used:

CF 07-42 (+) Fuse the aprE promoter to kudzuisoprene synthase gene (GTG start codon) (SEQ ID NO: 60)5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAAT CF 07-45 (−) Fuse the 3′end of kudzu isoprene synthase gene to the terminator (SEQ ID NO: 61)5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATC

c) Amplification of the Transcription Terminator

The terminator from the alkaline serine protease of Bacillusamyliquefaciens was amplified from a previously sequenced plasmidpJHPms382 using the following primers:

CF 07-44 (+) Fuse the 3′end of kudzu isoprene synthase to the terminator(SEQ ID NO: 62) 5′-GATTAACCAGCTGATGTATGTCTAAAAAAAACCGGCCTTGGCF 07-46 (−) End of B. amyliquefaciens  terminator (BamHI)(SEQ ID NO: 63) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The kudzu fragment was fused to the terminator fragment using PCR withthe following primers:

CF 07-42 (+) Fuse the aprE promoter to kudzuisoprene synthase gene (GTG start codon) (SEQ ID NO: 60)5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAATCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI) (SEQ ID NO:63)5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The kudzu-terminator fragment was fused to the promoter fragment usingPCR with the following primers:

CF 797 (+) Start aprE promoter MfeI (SEQ ID NO: 64)5′-GACATCAATTGCTCCATTTTCTTCTGCTATCCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI)(SEQ ID NO: 63) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The fusion PCR fragment was purified using a Qiagen kit and digestedwith the restriction enzymes MfeI and BamHI. This digested DNA fragmentwas gel purified using a Qiagen kit and ligated to a vector known aspBS19, which had been digested with EcoRI and BamHI and gel purified.

The ligation mix was transformed into E. coli Top 10 cells and colonieswere selected on LA+50 carbenicillin plates. A total of six colonieswere chosen and grown overnight in LB+50 carbenicillin and then plasmidswere isolated using a Qiagen kit. The plasmids were digested with EcoRIand BamHI to check for inserts and three of the correct plasmids weresent in for sequencing with the following primers:

CF 149 (+) EcoRI start of aprE promoter (SEQ ID NO: 65)5′-GACATGAATTCCTCCATTTTCTTCTGCCF 847 (+) Sequence in pXX 049 (end of aprE promoter) (SEQ ID NO: 66)5′-AGGAGAGGGTAAAGAGTGAG CF 07-45 (−) Fuse the 3′ end of kudzu isoprenesynthase to the terminator (SEQ ID NO: 61)5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATCCF 07-48 (+) Sequencing primer for kudzu isoprene synthase(SEQ ID NO: 67) 5′-CTTTTCCATCACCCACCTGAAGCF 07-49 (+) Sequencing in kudzu isoprene synthase (SEQ ID NO: 68)5′-GGCGAAATGGTCCAACAACAAAATTATC

The plasmid designated pBS Kudzu #2 (FIGS. 52 and 12) was correct bysequencing and was transformed into BG 3594 comK, a Bacillus subtilishost strain. Selection was done on LA+5 chloramphenicol plates. Atransformant was chosen and struck to single colonies on LA+5chloramphenicol, then grown in LB+5 chloramphenicol until it reached anOD₆₀₀ of 1.5. It was stored frozen in a vial at −80° C. in the presenceof glycerol. The resulting strain was designated CF 443.

II. Production of Isoprene in Shake Flasks Containing B. subtilis CellsExpressing Recombinant Isoprene Synthase

Overnight cultures were inoculated with a single colony of CF 443 from aLA+Chloramphenicol (Cm, 25 μg/ml). Cultures were grown in LB+Cm at 37°C. with shaking at 200 rpm. These overnight cultures (1 ml) were used toinoculate 250 ml baffled shake flasks containing 25 ml Grants II mediaand chloramphenicol at a final concentration of 25 μg/ml. Grants IIMedia recipe was 10 g soytone, 3 ml 1M K₂HPO₄, 75 g glucose, 3.6 g urea,100 ml 10×MOPS, q.s. to 1 L with H₂O, pH 7.2; 10×MOPS recipe was 83.72 gMOPS, 7.17 g tricine, 12 g KOH pellets, 10 ml 0.276M K₂SO₄ solution, 10ml 0.528M MgCl₂ solution, 29.22 g NaCl, 100 ml 100× micronutrients, q.s.to 1 L with H₂O; and 100× micronutrients recipe was 1.47 g CaCl₂*2H₂O,0.4 g FeSO₄.7H₂O, 0.1 g MnSO₄*H₂O, 0.1 g ZnSO₄*H₂O, 0.05 g CuCl₂*2H₂O,0.1 g CoCl₂*6H₂O, 0.1 g Na₂MoO₄*2H₂O, q.s. to 1 L with H₂O, Shake flaskswere incubated at 37° C. and samples were taken at 18, 24, and 44 hours.At 18 hours the headspaces of CF443 and the control strain were sampled.This represented 18 hours of accumulation of isoprene. The amount ofisoprene was determined by gas chromatography as described in Example 1.Production of isoprene was enhanced significantly by expressingrecombinant isoprene synthase (FIG. 11).

III. Production of Isoprene by CF443 in 14 L Fermentation

Large scale production of isoprene from B. subtilis containing therecombinant kudzu isoprene synthase gene on a replication plasmid wasdetermined from a fed-batch culture. Bacillus strain CF 443, expressinga kudzu isoprene synthase gene, or control stain which does not expressa kudzu isoprene synthase gene were cultivated by conventional fed-batchfermentation in a nutrient medium containing soy meal (Cargill), sodiumand potassium phosphate, magnesium sulfate and a solution of citricacid, ferric chloride and manganese chloride. Prior to fermentation themedia is macerated for 90 minutes using a mixture of enzymes includingcellulases, hemicellulases and pectinases (see, WO95/04134). 14-L batchfermentations are fed with 60% wt/wt glucose (Cargill DE99 dextrose, ADMVersadex greens or Danisco invert sugar) and 99% wt/wt oil (WesternFamily soy oil, where the 99% wt/wt is the concentration of oil beforeit was added to the cell culture medium). Feed was started when glucosein the batch was non-detectable. The feed rate was ramped over severalhours and was adjusted to add oil on an equal carbon basis. The pH wascontrolled at 6.8-7.4 using 28% w/v ammonium hydroxide. In case offoaming, antifoam agent was added to the media. The fermentationtemperature was controlled at 37° C. and the fermentation culture wasagitated at 750 rpm. Various other parameters such as pH, D0%, airflow,and pressure were monitored throughout the entire process. The DO % ismaintained above 20. Samples were taken over the time course of 36 hoursand analyzed for cell growth (OD₅₅₀) and isoprene production. Results ofthese experiments are presented in FIGS. 53A and 53B.

IV. Integration of the Kudzu Isoprene Synthase (ispS) in B. subtilis.

The kudzu isoprene synthase gene was cloned in an integrating plasmid(pJH101-cmpR) under the control of the aprE promoter. Under theconditions tested, no isoprene was detected.

Example 5 Production of Isoprene in Trichoderma

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein Trichoderma reesei

The Yarrowia lipolytica codon-optimized kudzu IS gene was synthesized byDNA 2.0 (SEQ ID NO:8) (FIG. 13). This plasmid served as the template forthe following PCR amplification reaction: 1 pJ plasmid template (20ng/ul), 1 pJ Primer EL-945 (10 uM)5′-GCTTATGGATCCTCTAGACTATTACACGTACATCAATTGG (SEQ ID NO:9), 1 μl PrimerEL-965 (10 uM) 5′-CACCATGTGTGCAACCTCCTCCCAGTTTAC (SEQ ID NO:10), 1 μldNTP (10 mM), 5 pJ 10×PfuUltra II Fusion HS DNA Polymerase Buffer, 1 pJPfuUltra II Fusion HS DNA Polymerase, 40 pJ water in a total reactionvolume of 50 μl. The forward primer contained an additional 4nucleotides at the 5′-end that did not correspond to the Y. lipolyticacodon-optimized kudzu isoprene synthase gene, but was required forcloning into the pENTR/D-TOPO vector. The reverse primer contained anadditional 21 nucleotides at the 5′-end that did not correspond to theY. lipolytica codon-optimized kudzu isoprene synthase gene, but wereinserted for cloning into other vector backbones. Using the MJ ResearchPTC-200 Thermocycler, the PCR reaction was performed as follows: 95° C.for 2 minutes (first cycle only), 95° C. for 30 seconds, 55° C. for 30seconds, 72° C. for 30 seconds (repeat for 27 cycles), 72° C. for 1minute after the last cycle. The PCR product was analyzed on a 1.2%E-gel to confirm successful amplification of the Y. lipolyticacodon-optimized kudzu isoprene synthase gene.

The PCR product was then cloned using the TOPO pENTR/D-TOPO Cloning Kitfollowing manufacturer's protocol: 1 μl PCR reaction, 1 μl Saltsolution, 1 μl TOPO pENTR/D-TOPO vector and 3 μl water in a totalreaction volume of 6 μl. The reaction was incubated at room temperaturefor 5 minutes. One microliter of TOPO reaction was transformed intoTOP10 chemically competent E. coli cells. The transformants wereselected on LA+50 μg/mlkanamycin plates. Several colonies were pickedand each was inoculated into a 5 ml tube containing LB+50 μg/mlkanamycin and the cultures grown overnight at 37° C. with shaking at 200rpm. Plasmids were isolated from the overnight culture tubes usingQIAprep Spin Miniprep Kit, following manufacturer's protocol. Severalplasmids were sequenced to verify that the DNA sequence was correct.

A single pENTR/D-TOPO plasmid, encoding a Y. lipolytica codon-optimizedkudzu isoprene synthase gene, was used for Gateway Cloning into acustom-made pTrex3 g vector. Construction of pTrex3 g is described in WO2005/001036 A2. The reaction was performed following manufacturer'sprotocol for the Gateway LR Clonase II Enzyme Mix Kit (Invitrogen): 1 μlY. lipolytica codon-optimized kudzu isoprene synthase gene pENTR/D-TOPOdonor vector, 1 μl pTrex3 g destination vector, 6 μl TE buffer, pH 8.0in a total reaction volume of 8 μl. The reaction was incubated at roomtemperature for 1 hour and then 1 μl proteinase K solution was added andthe incubation continued at 37° C. for 10 minutes. Then 1 μl of reactionwas transformed into TOP10 chemically competent E. coli cells. Thetransformants were selected on LA+50 μg/ml carbenicillin plates. Severalcolonies were picked and each was inoculated into a 5 ml tube containingLB+50 μg/ml carbenicillin and the cultures were grown overnight at 37°C. with shaking at 200 rpm. Plasmids were isolated from the overnightculture tubes using QIAprep Spin Miniprep Kit (Qiagen, Inc.), followingmanufacturer's protocol. Several plasmids were sequenced to verify thatthe DNA sequence was correct.

Biolistic transformation of Y. lipolytica codon-optimized kudzu isoprenesynthase pTrex3 g plasmid (FIG. 14) into a quad delete Trichodermareesei strain was performed using the Biolistic PDS-1000/HE ParticleDelivery System (see WO 2005/001036 A2). Isolation of stabletransformants and shake flask evaluation was performed using protocollisted in Example 11 of patent publication WO 2005/001036 A2.

II. Production of Isoprene in Recombinant Strains of T. reesei

One ml of 15 and 36 hour old cultures of isoprene synthase transformantsdescribed above were transferred to head space vials. The vials weresealed and incubated for 5 hours at 30° C. Head space gas was measuredand isoprene was identified by the method described in Example 1. Two ofthe transformants showed traces of isoprene. The amount of isoprenecould be increased by a 14 hour incubation. The two positive samplesshowed isoprene at levels of about 0.5 μg/L for the 14 hour incubation.The untransformed control showed no detectable levels of isoprene. Thisexperiment shows that T. reesei is capable of producing isoprene fromendogenous precursor when supplied with an exogenous isoprene synthase.

Example 6 Production of Isoprene in Yarrowia

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein Yarrowia lipolytica.

The starting point for the construction of vectors for the expression ofthe kudzu isoprene synthase gene in Yarrowia lipolytica was the vectorpSPZ1(MAP29Spb). The complete sequence of this vector (SEQ ID No:11) isshown in FIG. 15.

The following fragments were amplified by PCR using chromosomal DNA of aY. lipolytica strain GICC 120285 as the template: a promotorless form ofthe URA3 gene, a fragment of 18S ribosomal RNA gene, a transcriptionterminator of the Y. lipolytica XPR2 gene and two DNA fragmentscontaining the promoters of XPR2 and ICL1 genes. The following PCRprimers were used:

ICL1 3 (SEQ ID NO: 69) 5′-GGTGAATTCAGTCTACTGGGGATTCCCAAATCTATATATACTGCAGGTGAC ICL1 5 (SEQ ID NO: 70) 5′-GCAGGTGGGAAACTATGCACTCC XPR 3(SEQ ID NO: 71) 5′-CCTGAATTCTGTTGGATTGGAGGATTGGATAGTGGG XPR 5(SEQ ID NO: 72) 5′-GGTGTCGACGTACGGTCGAGCTTATTGACC XPRT3 (SEQ ID NO: 73)5′-GGTGGGCCCGCATTTTGCCACCTACAAGCCAG XPRT 5 (SEQ ID NO: 74)5′-GGTGAATTCTAGAGGATCCCAACGCTGTTGCCTACAACGG Y18S3 (SEQ ID NO: 75)5′-GGTGCGGCCGCTGTCTGGACCTGGTGAGTTTCCCCG Y18S 5 (SEQ ID NO: 76)5′-GGTGGGCCCATTAAATCAGTTATCGTTTATTTGATAG YURA3 (SEQ ID NO: 77)5′-GGTGACCAGCAAGTCCATGGGTGGTTTGATCATGG YURA 50 (SEQ ID NO: 78)5′-GGTGCGGCCGCCTTTGGAGTACGACTCCAACTATG YURA 51 (SEQ ID NO: 79)5′-GCGGCCGCAGACTAAATTTATTTCAGTCTCC

For PCR amplification the PfuUltraII polymerase (Stratagene),supplier-provided buffer and dNTPs, 2.5 μM primers and the indicatedtemplate DNA were used as per the manufacturer's instructions. Theamplification was done using the following cycle: 95° C. for 1 min; 34×(95° C. for 30 sec; 55° C. for 30 sec; 72° C. for 3 min) and 10 min at72° C. followed by a 4° C. incubation.

Synthetic DNA molecules encoding the kudzu isoprene synthase gene,codon-optimized for expression in Yarrowia, was obtained from DNA 2.0(FIG. 16; SEQ ID NO:12). Full detail of the construction scheme of theplasmids pYLA(KZ1) and pYLI(KZ1) carrying the synthetic kudzu isoprenesynthase gene under control of XPR2 and ICL1 promoters respectively ispresented in FIG. 18. Control plasmids in which a mating factor gene(MAP29) is inserted in place of an isoprene synthase gene were alsoconstructed (FIGS. 18E and 18F).

A similar cloning procedure can be used to express a poplar (Populusalba×Populus tremula) isoprene synthase gene. The sequence of the poplarisoprene is described in Miller B. et al. (2001) Planta 213, 483-487 andshown in FIG. 17 (SEQ ID NO:13). A construction scheme for thegeneration the plasmids pYLA(POP1) and pYLI(POP1) carrying syntheticpoplar isoprene synthase gene under control of XPR2 and ICL1 promotersrespectively is presented in FIGS. 18A and B.

II. Production of Isoprene by Recombinant Strains of Y. lipolytica

Vectors pYLA(KZ1), pYLI(KZ1), pYLA(MAP29) and pYLI(MAP29) were digestedwith SacII and used to transform the strain Y. lipolytica CLIB 122 by astandard lithium acetate/polyethylene glycol procedure to uridineprototrophy. Briefly, the yeast cells grown in YEPD (1% yeast extract,2% peptone, 2% glucose) overnight, were collected by centrifugation(4000 rpm, 10 min), washed once with sterile water and suspended in 0.1M lithium acetate, pH 6.0. Two hundred μl aliquots of the cellsuspension were mixed with linearized plasmid DNA solution (10-20 μg),incubated for 10 minutes at room temperature and mixed with 1 ml of 50%PEG 4000 in the same buffer. The suspensions were further incubated for1 hour at room temperature followed by a 2 minutes heat shock at 42° C.Cells were then plated on SC his leu plates (0.67% yeast nitrogen base,2% glucose, 100 mg/L each of leucine and histidine). Transformantsappeared after 3-4 days of incubation at 30° C.

Three isolates from the pYLA(KZ1) transformation, three isolates fromthe pYLI(KZ1) transformation, two isolates from the pYLA(MAP29)transformation and two isolates from the pYLI(MAP29) transformation weregrown for 24 hours in YEP7 medium (1% yeast extract, 2% peptone, pH 7.0)at 30° C. with shaking. Cells from 10 ml of culture were collected bycentrifugation, resuspended in 3 ml of fresh YEP7 and placed into 15 mlscrew cap vials. The vials were incubated overnight at room temperaturewith gentle (60 rpm) shaking. Isoprene content in the headspace of thesevials was analyzed by gas chromatography using mass-spectrometricdetector as described in Example 1. All transformants obtained withpYLA(KZ1) and pYLI(KZ1) produced readily detectable amounts of isoprene(0.5 μg/L to 1 μg/L, FIG. 20). No isoprene was detected in the headspaceof the control strains carrying phytase gene instead of an isoprenesynthase gene.

Example 7 Production of Isoprene in E. Coli Expressing Kudzu IsopreneSynthase and idi, or dxs, or idi and dxs

I. Construction of Vectors Encoding Kudzu Isoprene Synthase and idi, ordxs, or idi and dxs for the Production of Isoprene in E. colii) Construction of pTrcKudzuKan

The bla gene of pTrcKudzu (described in Example 1) was replaced with thegene conferring kanamycin resistance. To remove the bla gene, pTrcKudzuwas digested with BspHI, treated with Shrimp Alkaline Phosphatase (SAP),heat killed at 65° C., then end-filled with Klenow fragment and dNTPs.The 5 kbp large fragment was purified from an agarose gel and ligated tothe kan^(r) gene which had been PCR amplified from pCR-Blunt-II-TOPOusing primers MCM22 5′-GATCAAGCTTAACCGGAATTGCCAGCTG (SEQ ID NO:14) andMCM23 5′-GATCCGATCGTCAGAAGAACTCGTCAAGAAGGC (SEQ ID NO:15), digested withHindIII and Pvul, and end-filled. A transformant carrying a plasmidconferring kanamycin resistance (pTrcKudzuKan) was selected on LAcontaining kanamycin 50 μg/ml.

ii) Construction of pTrcKudzu yIDI Kan

pTrcKudzuKan was digested with PstI, treated with SAP, heat killed andgel purified. It was ligated to a PCR product encoding idi from S.cerevisiae with a synthetic RBS. The primers for PCR were NsiI-YIDI 1 F5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC (SEQ ID NO:16) and PstI-YIDI 1R 5′-CCTTCTGCAGGACGCGTTGTTATAGC (SEQ ID NO:17); and the template was S.cerevisiae genomic DNA. The PCR product was digested with NsiI and PstIand gel purified prior to ligation. The ligation mixture was transformedinto chemically competent TOP10 cells and selected on LA containing 50μg/ml kanamycin. Several transformants were isolated and sequenced andthe resulting plasmid was called pTrcKudzu-yIDI(kan) (FIGS. 34 and 35).

iii) Construction of pTrcKudzu DXS Kan

Plasmid pTrcKudzuKan was digested with PstI, treated with SAP, heatkilled and gel purified. It was ligated to a PCR product encoding dxsfrom E. coli with a synthetic RBS. The primers for PCR were MCM 135′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCC G (SEQ IDNO:18) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:19); andthe template was E. coli genomic DNA. The PCR product was digested withNsiI and PstI and gel purified prior to ligation. The resultingtransformation reaction was transformed into TOP10 cells and selected onLA with kanamycin 50 μg/ml. Several transformants were isolated andsequenced and the resulting plasmid was called pTrcKudzu-DXS(kan) (FIGS.36 and 37).

iv) Construction of pTrcKudzu-yIDI-dxs (kan)

pTrcKudzu-ylDI(kan) was digested with PstI, treated with SAP, heatkilled and gel purified. It was ligated to a PCR product encoding E.coli dxs with a synthetic RBS (primers MCM135′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCC G (SEQ IDNO:18) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:19);template TOP10 cells) which had been digested with NsiI and PstI and gelpurified. The final plasmid was called pTrcKudzu-yIDI-dxs (kan) (FIGS.21 and 22).

v) Construction of pCL PtrcKudzu

A fragment of DNA containing the promoter, structural gene andterminator from Example 1 above was digested from pTrcKudzu using Sspland gel purified. It was ligated to pCL1920 which had been digested withPvuII, treated with SAP and heat killed. The resulting ligation mixturewas transformed into TOP10 cells and selected in LA containingspectinomycin 50 μg/ml. Several clones were isolated and sequenced andtwo were selected. pCL PtrcKudzu and pCL PtrcKudzu (A3) have the insertin opposite orientations (FIGS. 38-41).

vi) Construction of pCL PtrcKudzu yIDI

The NsiI-PstI digested, gel purified, IDI PCR amplicon from (ii) abovewas ligated into pCL PtrcKudzu which had been digested with PstI,treated with SAP, and heat killed. The ligation mixture was transformedinto TOP10 cells and selected in LA containing spectinomycin 50 μg/ml.Several clones were isolated and sequenced and the resulting plasmid iscalled pCL PtrcKudzu yIDI (FIGS. 42 and 43).

vii) Construction of pCL PtrcKudzu DXS

The NsiI-PstI digested, gel purified, DXS PCR amplicon from (iii) abovewas ligated into pCL PtrcKudzu (A3) which had been digested with PstI,treated with SAP, and heat killed. The ligation mixture was transformedinto TOP10 cells and selected in LA containing spectinomycin 50 μg/ml.Several clones were isolated and sequenced and the resulting plasmid iscalled pCL PtrcKudzu DXS (FIGS. 44 and 45).

II. Measurement of Isoprene in Headspace from Cultures Expressing KudzuIsoprene Synthase, idi, and/or dxs at Different Copy Numbers.

Cultures of E. coli BL21(λDE3) previously transformed with plasmidspTrcKudzu(kan) (A), pTrcKudzu-ylDI kan (B), pTrcKudzu-DXS kan (C),pTrcKudzu-yIDI-DXS kan (D) were grown in LB kanamycin 50 μg/mL. Culturesof pCL PtrcKudzu (E), pCL PtrcKudzu, pCL PtrcKudzu-ylDI (F) and pCLPtrcKudzu-DXS (G) were grown in LB spectinomycin 50 μg/mL. Cultures wereinduced with 400 μM IPTG at time 0 (OD₆₀₀ approximately 0.5) and samplestaken for isoprene headspace measurement (see Example 1). Results areshown in FIG. 23A-23G.

Plasmid pTrcKudzu-yIDI-dxs (kan) was introduced into E. coli strain BL21by transformation. The resulting strain BL21/pTrc Kudzu IDI DXS wasgrown overnight in LB containing kanamycin (50 μg/ml) at 20° C. and usedto inoculate shake flasks of TM3 (13.6 g K₂PO₄, 13.6 g KH₂PO₄, 2.0 gMgSO₄.7H₂O), 2.0 g citric acid monohydrate, 0.3 g ferric ammoniumcitrate, 3.2 g (NH₄)₂SO₄, 0.2 g yeast extract, 1.0 ml 1000× ModifiedTrace Metal Solution, adjusted to pH 6.8 and q.s. to H₂O, and filtersterilized) containing 1% glucose. Flasks were incubated at 30° C. untilan OD₆₀₀ of 0.8 was reached, and then induced with 400 μM IPTG. Sampleswere taken at various times after induction and the amount of isoprenein the head space was measured as described in Example 1. Results areshown in FIG. 23H.

III. Production of Isoprene from Biomass in E. coli/pTrcKudzu yIDI DXS

The strain BL21 pTrcKudzuIDIDXS was tested for the ability to generateisoprene from three types of biomass; bagasse, corn stover and soft woodpulp with glucose as a control. Hydrolysates of the biomass wereprepared by enzymatic hydrolysis (Brown, L. and Torget, R., 1996, NRELstandard assay method Lap-009 “Enzymatic Saccharification ofLignocellulosic Biomass”) and used at a dilution based upon glucoseequivalents. In this example, glucose equivalents were equal to 1%glucose. A single colony from a plate freshly transformed cells of BL21(DE3) pTrcKudzu yIDI DXS (kan) was used to inoculate 5 ml of LB pluskanamycin (50 μg/ml). The culture was incubated overnight at 25° C. withshaking. The following day the overnight culture was diluted to an OD₆₀₀of 0.05 in 25 ml of TM3+0.2% YE+1% feedstock. The feedstock was cornstover, bagasse, or softwood pulp. Glucose was used as a positivecontrol and no glucose was used as a negative control. Cultures wereincubated at 30° C. with shaking at 180 rpm. The culture was monitoredfor OD₆₀₀ and when it reached an OD₆₀₀ of ˜0.8, cultures were analyzedat 1 and 3 hours for isoprene production as described in Example 1.Cultures are not induced. All cultures containing added feedstockproduce isoprene equivalent to those of the glucose positive control.Experiments were done in duplicate and are shown in FIG. 46.

IV. Production of Isoprene from Invert Sugar in E. coli/pTrcKudzuIDIDXS

A single colony from a plate freshly transformed cells of BL21(λDE3)/pTrcKudzu yIDI DXS (kan) was used to inoculate 5 mL ofLB+kanamycin (50 μg/ml). The culture was incubated overnight at 25° C.with shaking. The following day the overnight culture was diluted to anOD₆₀₀ of 0.05 in 25 ml of TM3+0.2% YE+1% feedstock. Feedstock wasglucose, inverted glucose or corn stover. The invert sugar feedstock(Danisco Invert Sugar) was prepared by enzymatically treating sucrosesyrup. AFEX corn stover was prepared as described below (Part V). Thecells were grown at 30° C. and the first sample was measured when thecultures reached an OD₆₀₀ ˜0.8-1.0 (0 hour). The cultures were analyzedfor growth as measured by OD₆₀₀ and for isoprene production as inExample 1 at 0, 1 and 3 hours. Results are shown in FIG. 47.

V. Preparation of Hydrolysate from AFEX Pretreated Corn Stover

AFEX pretreated corn stover was obtained from Michigan BiotechnologyInstitute. The pretreatment conditions were 60% moisture, 1:1 ammonialoading, and 90° C. for 30 minutes, then air dried. The moisture contentin the AFEX pretreated corn stover was 21.27%. The contents of glucanand xylan in the AFEX pretreated corn stover were 31.7% and 19.1% (drybasis), respectively. The saccharification process was as follows; 20 gof AFEX pretreated corn stover was added into a 500 ml flask with 5 mlof 1 M sodium citrate buffer pH 4.8, 2.25 ml of Accellerase 1000, 0.1 mlof Grindamyl H121 (Danisco xylanase product from Aspergillus niger forbread-making industry), and 72.65 ml of DI water. The flask was put inan orbital shaker and incubated at 50° C. for 96 hours. One sample wastaken from the shaker and analyzed using HPLC. The hydrolysate contained38.5 g/l of glucose, 21.8 g/l of xylose, and 10.3 g/l of oligomers ofglucose and/or xylose.

VI. The Effect of Yeast Extract on Isoprene Production in E. coli Grownin Fed-Batch Culture

Fermentation was performed at the 14-L scale as previously describedwith E. coli cells containing the pTrcKudzu yIDI DXS plasmid describedabove. Yeast extract (Bio Springer, Montreal, Quebec, Canada) was fed atan exponential rate. The total amount of yeast extract delivered to thefermentor was varied between 70-830 g during the 40 hour fermentation.Optical density of the fermentation broth was measured at a wavelengthof 550 nm. The final optical density within the fermentors wasproportional to the amount of yeast extract added (FIG. 48A). Theisoprene level in the off-gas from the fermentor was determined aspreviously described. The isoprene titer increased over the course ofthe fermentation (FIG. 48B). The amount of isoprene produced waslinearly proportional to the amount of fed yeast extract (FIG. 48C).

VII. Production of Isoprene in 500 L Fermentation of pTrcKudzu DXS yIDI

A 500 liter fermentation of E. coli cells with a kudzu isoprenesynthase, S. cerevisiae IDI, and E. coli DXS nucleic acids (E. coli BL21(λDE3) pTrc Kudzu dxs yidi) was used to produce isoprene. The levels ofisoprene varied from 50 to 300 μg/L over a time period of 15 hours. Onthe basis of the average isoprene concentrations, the average flowthrough the device and the extent of isoprene breakthrough, the amountof isoprene collected was calculated to be approximately 17 g.

VIII. Production of Isoprene in 500 L Fermentation of E. coli Grown inFed-Batch Culture

Medium Recipe (Per Liter Fermentation Medium):

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferricammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 withammonium gas (NH₃) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g,and antibiotic were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in DI H₂O,pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with0.22 micron filter.

Fermentation was performed in a 500-L bioreactor with E. coli cellscontaining the pTrcKudzu yIDI DXS plasmid. This experiment was carriedout to monitor isoprene formation from glucose and yeast extract at thedesired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was prepared in soytone-yeastextract-glucose medium. After the inoculum grew to OD 0.15, measured at550 nm, 20 ml was used to inoculate a bioreactor containing 2.5-Lsoytone-yeast extract-glucose medium. The 2.5-L bioreactor was grown at30° C. to OD 1.0 and 2.0-L was transferred to the 500-L bioreactor.

Yeast extract (Bio Springer, Montreal, Quebec, Canada) and glucose werefed at exponential rates. The total amount of glucose and yeast extractdelivered to the bioreactor during the 50 hour fermentation was 181.2 kgand 17.6 kg, respectively. The optical density within the bioreactorover time is shown in FIG. 49A. The isoprene level in the off-gas fromthe bioreactor was determined as previously described. The isoprenetiter increased over the course of the fermentation (FIG. 49B). Thetotal amount of isoprene produced during the 50 hour fermentation was55.1 g and the time course of production is shown in FIG. 49C.

Example 8 Production of Isoprene in E. coli Expressing Kudzu IsopreneSynthase and Recombinant Mevalonic Acid Pathway Genes I. Cloning theLower MVA Pathway

The strategy for cloning the lower mevalonic pathway was as follows.Four genes of the mevalonic acid biosynthesis pathway; mevalonate kinase(MVK), phosphomevalonate kinase (PMK), diphosphomevalonte decarboxylase(MVD) and isopentenyl diphosphate isomerase genes were amplified by PCRfrom S. cerevisiae chromosomal DNA and cloned individually into the pCRBluntII TOPO plasmid (Invitrogen). In some cases, the idi gene wasamplified from E. coli chromosomal DNA. The primers were designed suchthat an E. coli consensus RBS (AGGAGGT (SEQ ID NO:80) or AAGGAGG (SEQ IDNO:81)) was inserted at the 5′ end, 8 bp upstream of the start codon anda PstI site was added at the 3′ end. The genes were then cloned one byone into the pTrcHis2B vector until the entire pathway was assembled.

Chromosomal DNA from S. cerevisiae S288C was obtained from ATCC (ATCC204508D). The MVK gene was amplified from the chromosome of S.cerevisiae using primers MVKF(5′-AGGAGGTAAAAAAACATGTCATTACCGTTCTTAACTTCTGC, SEQ ID NO:21) andMVK-Pst1-R (5′-ATGGCTGCAGGCCTATCGCAAATTAGCTTATGAAGTCCATGGTAAATTCGTG, SEQID NO:22) using PfuTurbo as per manufacturer's instructions. The correctsized PCR product (1370 bp) was identified by electrophoresis through a1.2% E-gel (Invitrogen) and cloned into pZeroBLUNT TOPO. The resultingplasmid was designated pMVK1. The plasmid pMVK1 was digested with SacIand Taq1 restriction endonucleases and the fragment was gel purified andligated into pTrcHis2B digested with SacI and BstBI. The resultingplasmid was named pTrcMVK1.

The second gene in the mevalonic acid biosynthesis pathway, PMK, wasamplified by PCR using primers: PstI-PMK1 R (5′-GAATTCGCCCTTCTGCAGCTACC,SEQ ID NO:23) and BsiHKA I-PMK1 F(5′-CGACTGGTGCACCCTTAAGGAGGAAAAAAACATGTCAG, SEQ ID NO:24). The PCRreaction was performed using Pfu Turbo polymerase (Stratagene) as permanufacturer's instructions. The correct sized product (1387 bp) wasdigested with PstI and BsiHKI and ligated into pTrcMVK1 digested withPstI. The resulting plasmid was named pTrcKK. The MVD and the idi geneswere cloned in the same manner. PCR was carried out using the primerpairs PstI-MVD 1 R (5′-GTGCTGGAATTCGCCCTTCTGCAGC, SEQ ID NO:25) andNsiI-MVD 1 F (5′-GTAGATGCATGCAGAATTCGCCCTTAAGGAGG, SEQ ID NO:26) toamplify the MVD gene and PstI-YIDI 1 R (5′-CCTTCTGCAGGACGCGTTGTTATAGC,SEQ ID NO:27) and NsiI-YIDI 1 F(5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC, SEQ ID NO:28) to amplify theyIDI gene. In some cases the IPP isomerase gene, idi from E. coli wasused. To amplify idi from E. coli chromosomal DNA, the following primerset was used: PstI-CIDI 1 R (5′-GTGTGATGGATATCTGCAGAATTCG, SEQ ID NO:29)and NsiI-CIDI 1 F (5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAACATG, SEQ IDNO:30). Template DNA was chromosomal DNA isolated by standard methodsfrom E. coli FM5 (WO 96/35796 and WO 2004/033646, which are each herebyincorporated by reference in their entireties, particularly with respectto isolation of nucleic acids). The final plasmids were named pKKDIy forthe construct encoding the yeast idi gene or pKKDIc for the constructencoding the E. coli idi gene. The plasmids were transformed into E.coli hosts BL21 for subsequent analysis. In some cases the isoprenesynthase from kudzu was cloned into pKKDIy yielding plasmid pKKDIyIS.

The lower MVA pathway was also cloned into pTrc containing a kanamycinantibiotic resistance marker. The plasmid pTrcKKDIy was digested withrestriction endonucleases ApaI and PstI, the 5930 bp fragment wasseparated on a 1.2% agarose E-gel and purified using the Qiagen GelPurification kit according to the manufacturer's instructions. Theplasmid pTrcKudzuKan, described in Example 7, was digested withrestriction endonucleases ApaI and PstI, and the 3338 bp fragmentcontaining the vector was purified from a 1.2% E-gel using the QiagenGel Purification kit. The 3338 bp vector fragment and the 5930 bp lowerMVA pathway fragment were ligated using the Roche Quick Ligation kit.The ligation mix was transformed into E. coli TOP10 cells andtranformants were grown at 37° C. overnight with selection on LAcontaining kanamycin (50 μg/ml). The transformants were verified byrestriction enzyme digestion and one was frozen as a stock. The plasmidwas designated pTrcKanKKDIy.

II. Cloning a Kudzu Isoprene Synthase Gene into pTrcKanKKDIy

The kudzu isoprene synthase gene was amplified by PCR from pTrcKudzu,described in Example 1, using primers MCM505′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGTGTGCGACCTCTTCTCAATTTAC T (SEQ IDNO:31) and MCM53 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ IDNO:32). The resulting PCR fragment was cloned into pCR2.1 andtransformed into E. coli TOP10. This fragment contains the codingsequence for kudzu isoprene synthase and an upstream region containing aRBS from E. coli. Transformants were incubated overnight at 37° C. withselection on LA containing carbenicillin (50 μg/ml). The correctinsertion of the fragment was verified by sequencing and this strain wasdesignated MCM93.

The plasmid from strain MCM93 was digested with restrictionendonucleases NsiI and PstI to liberate a 1724 bp insert containing theRBS and kudzu isoprene synthase. The 1724 bp fragment was separated on a1.2% agarose E-gel and purified using the Qiagen Gel Purification kitaccording to the manufacturer's instructions. Plasmid pTrcKanKKDIy wasdigested with the restriction endonuclease PstI, treated with SAP for 30minutes at 37° C. and purified using the Qiagen PCR cleanup kit. Theplasmid and kudzu isoprene synthase encoding DNA fragment were ligatedusing the Roche Quick Ligation kit. The ligation mix was transformedinto E. coli TOP10 cells and transformants were grown overnight at 37°C. with selection on LA containing Kanamycin at 50 μg/ml. The correcttransformant was verified by restriction digestion and the plasmid wasdesignated pTrcKKDyIkISKan (FIGS. 24 and 25). This plasmid wastransformed into BL21(λDE3) cells (Invitrogen).

III. Isoprene Production from Mevalonate in E. coli Expressing theRecombinant Lower Mevalonate Pathway and Isoprene Synthase from Kudzu.

Strain BL21/pTrcKKDyIkISKan was cultured in MOPS medium (Neidhardt etal., (1974) J. Bacteriology 119:736-747) adjusted to pH 7.1 andsupplemented with 0.5% glucose and 0.5% mevalonic acid. A controlculture was also set up using identical conditions but without theaddition of 0.5% mevalonic acid. The culture was started from anovernight seed culture with a 1% inoculum and induced with 500 μM IPTGwhen the culture had reached an OD₆₀₀ of 0.3 to 0.5. The cultures weregrown at 30° C. with shaking at 250 rpm. The production of isoprene wasanalyzed 3 hours after induction by using the head space assay describedin Example 1. Maximum production of isoprene was 6.67×10⁻⁴mol/L_(broth)/OD₆₀₀/hr where L_(broth) is the volume of broth andincludes both the volume of the cell medium and the volume of the cells.The control culture not supplemented with mevalonic acid did not producemeasurable isoprene.

IV. Cloning the Upper MVA Pathway

The upper mevalonate biosynthetic pathway, comprising two genes encodingthree enzymatic activities, was cloned from Enterococcus faecalis. ThemvaE gene encodes a protein with the enzymatic activities of bothacetyl-CoA acetyltransferase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase, the first and third proteins in the pathway, andthe mvaS gene encodes second enzyme in the pathway, HMG-CoA synthase.The mvaE gene was amplified from E. faecalis genomic DNA (ATCC700802D-5) with an E. coli ribosome binding site and a spacer in frontusing the following primers:

CF 07-60 (+) Start of mvaE w/RBS + ATG start codon SacI (SEQ ID NO: 34)5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGT AGTTATTATTGCF 07-62 (−) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 35)5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTA TTGTTTTCTTAAATC

The mvaS gene was amplified from E. faecalis genomic DNA (ATCC700802D-5) with a RBS and spacer from E. coli in front using thefollowing primers:

CF 07-61 (+) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 36)5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACA ATTGGGATTGATAAACF 07-102 (−) End of mvaS gene BgIII (SEQ ID NO: 37)5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT

The PCR fragments were fused together with PCR using the followingprimers:

CF 07-60 (+) Start of mvaE w/RBS + ATG start codon SacI (SEQ ID NO: 34)5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAG TAGTTATTATTGCF 07-102 (−) End of mvaS gene BgIII (SEQ ID NO: 37)5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT

The fusion PCR fragment was purified using a Qiagen kit and digestedwith the restriction enzymes SacI and BglII. This digested DNA fragmentwas gel purified using a Qiagen kit and ligated into the commerciallyavailable vector pTrcHis2A, which had been digested with Sad and BglIIand gel purified.

The ligation mix was transformed into E. coli Top 10 cells and colonieswere selected on LA+50 μg/ml carbenicillin plates. A total of sixcolonies were chosen and grown overnight in LB+50 μg/ml carbenicillinand plasmids were isolated using a Qiagen kit. The plasmids weredigested with SacI and BglII to check for inserts and one correctplasmid was sequenced with the following primers:

CF 07-58 (+) Start of mvaE gene (SEQ ID NO: 38)5′-ATGAAAACAGTAGTTATTATTGATGC CF 07-59 (−) End of mvaE gene(SEQ ID NO: 39) 5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGCCF 07-82 (+) Start of mvaS gene (SEQ ID NO: 40)5′ATGACAATTGGGATTGATAAAATTAG CF 07-83 (−) End of mvaS gene(SEQ ID NO: 41) 5′-TTAGTTTCGATAAGAACGAACGGTCF 07-86 (+) Sequence in mvaE (SEQ ID NO: 42) 5′-GAAATAGCCCCATTAGAAGTATCCF 07-87 (+) Sequence in mvaE (SEQ ID NO: 43)5′-TTGCCAATCATATGATTGAAAATC CF 07-88 (+) Sequence in mvaE(SEQ ID NO: 44) 5′-GCTATGCTTCATTAGATCCTTATCG CF 07-89 (+) Sequence mvaS(SEQ ID NO: 45) 5′-GAAACCTACATCCAATCTTTTGCCC

The plasmid called pTrcHis2AUpperPathway#1 was correct by sequencing andwas transformed into the commercially available E. coli strain BL21.Selection was done on LA+50 μg/ml carbenicillin. Two transformants werechosen and grown in LB+50 μg/mlcarbenicillin until they reached an OD₆₀₀of 1.5. Both strains were frozen in a vial at −80° C. in the presence ofglycerol. Strains were designated CF 449 for pTrcHis2AUpperPathway#1 inBL21, isolate #1 and CF 450 for pTrcHis2AUpperPathway#1 in BL21, isolate#2. Both clones were found to behave identically when analyzed.

V. Cloning of UpperMVA Pathway into pCL1920

The plasmid pTrcHis2AUpperPathway was digested with the restrictionendonuclease Sspl to release a fragment containing pTrc-mvaE-mvaS-(Histag)-terminator. In this fragment, the his-tag was not translated. Thisblunt ended 4.5 kbp fragment was purified from a 1.2% E-gel using theQiagen Gel Purification kit. A dephosphorylated, blunt ended 4.2 kbpfragment from pCL1920 was prepared by digesting the vector with therestriction endonuclease PvuII, treating with SAP and gel purifying froma 1.2% E-gel using the Qiagen Gel Purification kit. The two fragmentswere ligated using the Roche Quick Ligation Kit and transformed intoTOP10 chemically competent cells. Transformants were selected on LAcontaining spectinomycin (50 μg/ml). A correct colony was identified byscreening for the presence of the insert by PCR. The plasmid wasdesignated pCL PtrcUpperPathway (FIGS. 26 and 27A-27D).

VI. Strains Expressing the Combined Upper and Lower Mevalonic AcidPathways

To obtain a strain with a complete mevalonic acid pathway plus kudzuisoprene synthase, plasmids pTrcKKDyIkISkan and pCLpTrcUpperPathway wereboth transformed into BL21(λDE3) competent cells (Invitrogen) andtransformants were selected on LA containing kanamycin (50 μg/ml) andSpectinomycin (50 μg/ml). The transformants were checked by plasmid prepto ensure that both plasmids were retained in the host. The strain wasdesignated MCM127.

VII. Production of Mevalonic Acid from Glucose in E. coli/pUpperpathway

Single colonies of the BL21/pTrcHis2A-mvaE/mvaS or FM5/ppTrcHis2A-mvaE/mvaS are inoculated into LB+carbenicillin (100 μg/ml) andare grown overnight at 37° C. with shaking at 200 rpm. These cultureswere diluted into 50 ml medium in 250 ml baffled flasks to an OD₆₀₀ of0.1. The medium was TM3+1 or 2% glucose+carbenicillin (100 ug/ml) orTM3+1% glucose+hydrolyzed soy oil+carbenicillin (100 ug/ml) orTM3+biomass (prepared bagasse, corn stover or switchgrass). Cultureswere grown at 30° C. with shaking at 200 rpm for approximately 2-3 hoursuntil an OD₆₀₀ of 0.4 was reached. At this point the expression from themvaE mvaS construct was induced by the addition of IPTG (400 μM).Cultures were incubated for a further 20 or 40 hours with samples takenat 2 hour intervals to 6 hour post induction and then at 24, 36 and 48hours as needed. Sampling was done by removing 1 ml of culture,measuring the OD₆₀₀, pelleting the cells in a microfuge, removing thesupernatant and analyzing it for mevalonic acid.

A 14 liter fermentation of E. coli cells with nucleic acids encodingEnterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoAreductase polypeptides produced 22 grams of mevalonic acid with TM3medium and 2% glucose as the cell medium. A shake flask of these cellsproduced 2-4 grams of mevalonic acid per liter with LB medium and 1%glucose as the cell culture medium. The production of mevalonic acid inthese strains indicated that the MVA pathway was functional in E. coli.

VIII. Production of Isoprene from E. coli BL21 Containing the Upper andLower MVA Pathway Plus Kudzu Isoprene Synthase.

The following strains were created by transforming in variouscombinations of plasmids containing the upper and lower MVA pathway andthe kudzu isoprene synthase gene as described above and the plasmidscontaining the idi, dxs, and dxr and isoprene synthase genes describedin Example 7. The host cells used were chemically competent BL21(λDE3)and the transformations were done by standard methods. Transformantswere selected on L agar containing kanamycin (50 μg/ml) or kanamycinplus spectinomycin (both at a concentration of 50 μg/ml). Plates weregrown at 37° C. The resulting strains were designated as follows:

Grown on Kanamycin plus Spectinomycin (50 μg/ml each)MCM127-pCL Upper MVA+pTrcKKDyIkIS (kan) in BL21(λDE3)MCM131-pCL1920+pTrcKKDyIkIS (kan) in BL21(λDE3)MCM125-pCL Upper MVA+pTrcHis2B (kan) in BL21(λDE3)Grown on Kanamycin (50 μg/ml)MCM64-pTrcKudzu yIDI DXS (kan) in BL21(λDE3)MCM50-pTrcKudzu (kan) in BL21(λDE3)MCM123-pTrcKudzu yIDI DXS DXR (kan) in BL21(λDE3)

The above strains were streaked from freezer stocks to LA+appropriateantibiotic and grown overnight at 37° C. A single colony from each platewas used to inoculate shake flasks (25 ml LB+the appropriateantibiotic). The flasks were incubated at 22° C. overnight with shakingat 200 rpm. The next morning the flasks were transferred to a 37° C.incubator and grown for a further 4.5 hours with shaking at 200 rpm. The25 ml cultures were centrifuged to pellet the cells and the cells wereresuspended in 5 ml LB+the appropriate antibiotic. The cultures werethen diluted into 25 ml LB+1% glucose+the appropriate antibiotic to anOD₆₀₀ of 0.1. Two flasks for each strain were set up, one set forinduction with IPTG (800 μM) the second set was not induced. Thecultures were incubated at 37° C. with shaking at 250 rpm. One set ofthe cultures were induced after 1.50 hours (immediately followingsampling time point 1). At each sampling time point, the OD₆₀₀ wasmeasured and the amount of isoprene determined as described inExample 1. Results are presented in Table 3. The amount of isoprene madeis presented as the amount at the peak production for the particularstrain.

TABLE 3 Production of isoprene in E. coli strains Strain Isoprene(μg/liter/OD/hr MCM50 23.8 MCM64 289 MCM125 ND MCM131 Trace MCM127 874ND: not detected Trace: peak present but not integrable. IX. Analysis ofmevalonic acid

Mevalonolactone (1.0 g, 7.7 mmol) (CAS#503-48-0) was supplied fromSigma-Aldrich (WI, USA) as a syrup that was dissolved in water (7.7 mL)and was treated with potassium hydroxide (7.7 mmol) in order to generatethe potassium salt of mevalonic acid. The conversion to mevalonic acidwas confirmed by ¹H NMR analysis. Samples for HPLC analysis wereprepared by centrifugation at 14,000 rpm for 5 minutes to remove cells,followed by the addition of a 300 μl aliquot of supernatant to 900 μl ofH₂O. Perchloric acid (36 μl of a 70% solution) was then added followedby mixing and cooling on ice for 5 minutes. The samples were thencentrifuged again (14,000 rpm for 5 min) and the supernatant transferredto HPLC. Mevalonic acid standards (20, 10, 5, 1 and 0.5 g/L) wereprepared in the same fashion. Analysis of mevalonic acid (20 uLinjection volume) was performed by HPLC using a BioRad Aminex 87-H+column (300 mm by 7.0 mm) eluted with 5 mM sulfuric acid at 0.6 mL/minwith refractive index (R1) detection. Under these conditions mevalonicacid eluted as the lactone form at 18.5 minutes.

X. Production of Isoprene from E. coli BL21 Containing the Upper MVAPathway Plus Kudzu Isoprene Synthase

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides and Kudzu isoprene synthase was used to produce isoprenefrom cells in fed-batch culture. This experiment demonstrates thatgrowing cells under glucose limiting conditions resulted in theproduction of 2.2 g/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCL PtrcUpperPathway (FIG. 26) and pTrcKKDyIkISplasmids. This experiment was carried out to monitor isoprene formationfrom glucose at the desired fermentation pH 7.0 and temperature 30° C.An inoculum of E. coli strain taken from a frozen vial was streaked ontoan LB broth agar plate (with antibiotics) and incubated at 37° C. Asingle colony was inoculated into soytone-yeast extract-glucose medium.After the inoculum grew to OD 1.0 when measured at 550 nm, 500 mL wasused to inoculate a 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 54 hour fermentation was 3.7 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 25 uM when the optical density at 550nm (OD₅₅₀) reached a value of 10. The IPTG concentration was raised to50 uM when OD₅₅₀ reached 190. IPTG concentration was raised to 100 uM at38 hours of fermentation. The OD₅₅₀ profile within the bioreactor overtime is shown in FIG. 54. The isoprene level in the off gas from thebioreactor was determined as described herein. The isoprene titerincreased over the course of the fermentation to a final value of 2.2g/L (FIG. 55). The total amount of isoprene produced during the 54 hourfermentation was 15.9 g, and the time course of production is shown inFIG. 56.

XI. Isoprene Fermentation from E. coli Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides and Kudzu isoprene synthase was used to produce isoprenefrom cells in fed-batch culture. This experiment demonstrates thatgrowing cells under glucose limiting conditions resulted in theproduction of 3.0 g/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× Modified Trace Metal Solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-Lbioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time, the glucose feed was decreased tomeet metabolic demands. The total amount of glucose delivered to thebioreactor during the 59 hour fermentation was 2.2 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 uMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 10. TheIPTG concentration was raised to 50 uM when OD₅₅₀ reached 190. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 93. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 3.0 g/L (FIG. 94). The total amount ofisoprene produced during the 59 hour fermentation was 22.8 g, and thetime course of production is shown in FIG. 95. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 2.2%. The weight percent yield of isoprene from glucose was 1.0%.

XII. Isoprene Fermentation from E. coli Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides, Pueraria lobata isoprene synthase, and Kudzu isoprenesynthase was used to produce isoprene from cells in fed-batch culture.This experiment demonstrates that growing cells under glucose limitingconditions resulted in the production of 3.3 g/L of isoprene.

i) Construction of pCLPtrcUpperPathwayHGS2

The gene encoding isoprene synthase from Pueraria lobata wasPCR-amplified using primers NsiI-RBS-HGS F(CTTGATGCATCCTGCATTCGCCCTTAGGAGG, SEQ ID NO:88) and pTrcR(CCAGGCAAATTCTGTTTTATCAG, SEQ ID NO:89), and pTrcKKDyIkIS as a template.The PCR product thus obtained was restriction-digested with NsiI andPstI and gel-purified. The plasmid pCL PtrcUpperPathway wasrestriction-digested with PstI and dephosphorylated using rAPid alkalinephosphatase (Roche) according to manufacturer's instructions.

These DNA fragments were ligated together and the ligation reaction wastransformed into E. coli Top10 chemically competent cells (Invitrogen),plated on L agar containing spectinomycin (50 ug/ml) and incubatedovernight at 370 C. Plasmid DNA was prepared from 6 clones using theQiaquick Spin Mini-prep kit. The plasmid DNA was digested withrestriction enzymes EcoRV and M/uI to identify a clone in which theinsert had the right orientation (i.e., the gene oriented in the sameway as the pTrc promoter).

The resulting correct plasmid was designated pCLPtrcUpperPathwayHGS2.This plasmid was assayed using the headspace assay described herein andfound to produce isoprene in E. coli Top10, thus validating thefunctionality of the gene. The plasmid was transformed into BL21(LDE3)containing pTrcKKDyIkIS to yield the strainBL21/pCLPtrcUpperPathwayHGS2-pTrcKKDyIkIS. This strain has an extra copyof the isoprene synthase compared to the BL21/pCL PtrcUpperMVA and pTrcKKDyIkIS strain (Example 8, part XI). This strain also had increasedexpression and activity of HMGS compared to the BL21/pCL PtrcUpperMVAand pTrc KKDyIkIS strain used in Example 8, part XI.

ii) Isoprene Fermentation from E. coli ExpressingpCLPtrcUpperPathwayHGS2-pTrcKKDyIkIS and Grown in Fed-Batch Culture atthe 15-L Scale

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCLPtrcUpperPathwayHGS2 and pTrc KKDyIkIS plasmids.This experiment was carried out to monitor isoprene formation fromglucose at the desired fermentation pH 7.0 and temperature 30° C. Aninoculum of E. coli strain taken from a frozen vial was streaked onto anLB broth agar plate (with antibiotics) and incubated at 37° C. A singlecolony was inoculated into tryptone-yeast extract medium. After theinoculum grew to OD 1.0 measured at 550 nm, 500 mL was used to inoculatea 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 58 hour fermentation was 2.1 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 uMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 9. TheIPTG concentration was raised to 50 uM when OD₅₅₀ reached 170. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 104. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 3.3 g/L (FIG. 105). The total amount ofisoprene produced during the 58 hour fermentation was 24.5 g and thetime course of production is shown in FIG. 106. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 2.5%. The weight percent yield of isoprene from glucose was 1.2%.Analysis showed that the activity of the isoprene synthase was increasedby approximately 3-4 times that compared to BL21 expressing CLPtrcUpperMVA and pTrc KKDyIkIS plasmids (data not shown).

XIII Chromosomal Integration of the Lower Mevalonate Pathway in E. coli.

A synthetic operon containing mevalonate kinase, mevalonate phosphatekinase, mevalonate pyrophosphate decarboxylase, and the IPP isomerasewas integerated into the chromosome of E. coli. If desired, expressionmay be altered by integrating different promoters 5′ of the operon.

Table 9 lists primers used for this experiment.

TABLE 9 Primers MCM78 attTn7 up rev forgcatgctcgagcggccgcTTTTAATCAAACATCC integration constructTGCCAACTC (SEQ ID NO: 91) MCM79 attTn7 down rev forgatcgaagggcgatcgTGTCACAGTCTGGCGAAA integration constructCCG (SEQ ID NO: 92) MCM88 attTn7 up forw forctgaattctgcagatatcTGTTTTTCCACTCTTC integration constructGTTCACTTT (SEQ ID NO: 93) MCM89 attTn7 down forw fortctagagggcccAAGAAAAATGCCCCGCTTACG integration construct (SEQ ID NO: 94)MCM104 GI1.2 promoter - MVK Gatcgcggccgcgcccttgacgatgccacatcctgagcaaataattcaaccactaattgtgagcggat aacacaaggaggaaacagctatgtcattaccgttcttaacttc (SEQ ID NO: 95) MCM105 aspA terminator - yIDIGatcgggccccaagaaaaaaggcacgtcatctgac gtgccttttttatttgtagacgcgttgttatagcattcta (SEQ ID NO: 96) MCM120 Forward of attTn7:aaagtagccgaagatgacggtttgtcacatggagt attTn7 homology, GBtggcaggatgtttgattaaaagcAATTAACCCTCA marker homologyCTAAAGGGCGG (SEQ ID NO: 97) MCM127 Rev complement of 1.2  AGAGTGTTCACCAAAAATAATAACCTTTCCCGGTG GI: GB marker homologyCAgaagttaagaacggtaatgacatagctgtttcc   (extra long), promoter,tccttgtgttatccgctcacaattagtggttgaat RBS, ATGtatttgctcaggatgtggcatcgtcaagggcTAAT ACGACTCACTATAGGGCTCG (SEQ ID NO: 98)

i) Target Vector Construction

The attTn7 site was selected for integration. Regions of homologyupstream (attTn7 up) (primers MCM78 and MCM79) and downstream (attTn7down) (primers MCM88 and MCM89) were amplified by PCR from MG1655 cells.A 50 uL reaction with luL 10 uM primers, 3 uL ddH2O, 45 uL InvitrogenPlatinum PCR Supermix High Fidelity, and a scraped colony of MG1655 wasdenatured for 2:00 at 940 C, cycled 25 times (2:00 at 940 C, 0:30 at 500C, and 1:00 at 680 C), extended for 7:00 at 720 C, and cooled to 40 C.This resulting DNA was cloned into pCR2.1 (Invitrogen) according to themanufacturer's instructions, resulting in plasmids MCM278 (attTn7 up)and MCM252 (attTn7 down). The 832 bp ApaI-Pvul fragment digested and gelpurified from MCM252 was cloned into ApaI-Pvul digested and gel purifiedplasmid pR6K, creating plasmid MCM276. The 825 bp PstI-NotI fragmentdigested and gel purified from MCM278 was cloned into PstI-NotI digestedand gel purified MCM276, creating plasmid MCM281.

ii) Cloning of Lower Pathway and Promoter

MVK-PMK-MVD-IDI genes were amplified from pTrcKKDyIkIS with primersMCM104 and MCM105 using Roche Expand Long PCR System according to themanufacturer's instructions. This product was digested with NotI andApaI and cloned into MCM281 which had been digested with NotI and ApaIand gel purified. Primers MCM 120 and MCM 127 were used to amplify CMRcassette from the GeneBridges FRT-gb2-Cm-FRT template DNA usingStratagene Pfu Ultra II. A PCR program of denaturing at 950 C for 4:00,5 cycles of 950 C for 0:20, 550 C for 0:20, 720 C for 2:00, 25 cycles of950 C for 0:20, 580 C for 0:20, 720 C for 2:00, 720 C for 10:00, andthen cooling to 40 C was used with four 50 uL PCR reactions containing 1uL˜10 ng/uL template, 1 uL each primer, 1.25 uL 10 mM dNTPs, 5 uL 10×buffer, luL enzyme, and 39.75 uL ddH20. Reactions were pooled, purifiedon a Qiagen PCR cleanup column, and used to electroporate water-washedPir1 cells containing plasmid MCM296. Electroporation was carried out in2 mM cuvettes at 2.5V and 200 ohms. Electroporation reactions wererecovered in LB for 3 hr at 300 C. Transformant MCM330 was selected onLA with CMPS, Kan50 (FIGS. 107 and 108A-108C).

iii) Integration into E. coli Chromosome

Miniprepped DNA (Qiaquick Spin kit) from MCM330 was digested with SnaBIand used to electroporate BL21(DE3) (Novagen) or MG1655 containingGeneBridges plasmid pRedET Carb. Cells were grown at 300 C to ˜OD1 theninduced with 0.4% L-arabinose at 370 C for 1.5 hours. These cells werewashed three times in 40 C ddH2O before electroporation with 2 uL ofDNA. Integrants were selected on L agar with containing chloramphenicol(5 ug/ml) and subsequently confirmed to not grow on L agar+Kanamycin (50ug/ml). BL21 integrant MCM331 and MG1655 integrant MCM333 were frozen.

iv) Construction of pET24D-Kudzu Encoding Kudzu Isoprene Synthase

The kudzu isoprene synthase gene was subcloned into the pET24d vector(Novagen) from the pCR2.1 vector (Invitrogen). In particular, the kudzuisoprene synthase gene was amplified from the pTrcKudzu template DNAusing primers MCM50 5′-GATCATGCAT TCGCCCTTAG GAGGTAAAAA AACATGTGTGCGACCTCTTC TCAATTTACT (SEQ ID NO:99) and MCM53 5′-CGGTCGACGG ATCCCTGCAGTTAGACATAC ATCAGCTG (SEQ ID NO:100). PCR reactions were carried outusing Taq DNA Polymerase (Invitrogen), and the resulting PCR product wascloned into pCR2.1-TOPO TA cloning vector (Invitrogen), and transformedinto E. coli Top10 chemically competent cells (Invitrogen).Transformants were plated on L agar containing carbenicillin (50 μg/ml)and incubated overnight at 37° C. Five ml Luria Broth culturescontaining carbenicillin 50 μg/ml were inoculated with singletransformants and grown overnight at 37° C. Five colonies were screenedfor the correct insert by sequencing of plasmid DNA isolated from 1 mlof liquid culture (Luria Broth) and purified using the QIAprep SpinMini-prep Kit (Qiagen). The resulting plasmid, designated MCM93,contains the kudzu isoprene synthase coding sequence in a pCR2.1backbone.

The kudzu coding sequence was removed by restriction endonucleasedigestion with PciI and BamH1 (Roche) and gel purified using theQIAquick Gel Extraction kit (Qiagen). The pET24d vector DNA was digestedwith NcoI and BamHI (Roche), treated with shrimp alkaline phosphatase(Roche), and purified using the QIAprep Spin Mini-prep Kit (Qiagen). Thekudzu isoprene synthase fragment was ligated to the NcoI/BamH1 digestedpET24d using the Rapid DNA Ligation Kit (Roche) at a 5:1 fragment tovector ratio in a total volume of 20 μl. A portion of the ligationmixture (5 μl) was transformed into E. coli Top 10 chemically competentcells and plated on L agar containing kanamycin (50 μg/ml). The correcttransformant was confirmed by sequencing and transformed into chemicallycompetent BL21(λDE3)pLysS cells (Novagen). A single colony was selectedafter overnight growth at 37° C. on L agar containing kanamycin (50μg/ml). A map of the resulting plasmid designated as pET24D-Kudzu isshown in FIG. 109. The sequence of pET24D-Kudzu (SEQ ID NO:101) is shownin FIGS. 110A and 110B. Isoprene synthase activity was confirmed using aheadspace assay.

v) Production Strains

Strains MCM331 and MCM333 were cotransformed with plasmidspCLPtrcupperpathway and either pTrcKudzu or pETKudzu, resulting in thestrains shown in Table 10.

TABLE 10 Production Strains Isoprene Integrated Upper MVA synthaseProduction Background Lower plasmid plasmid Stain BL21(DE3) MCM331pCLPtrcUpper pTrcKudzu MCM343 Pathway BL21(DE3) MCM331 pCLPtrcUpperpET24D- MCM335 Pathway Kudzu MG1655 MCM333 pCLPtrcUpper pTrcKudzu MCM345Pathwayvi) Isoprene Fermentation from E. COLI Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH2O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the gi1.2 integrated lower MVA pathway described aboveand the pCL PtrcUpperMVA and pTrcKudzu plasmids. This experiment wascarried out to monitor isoprene formation from glucose at the desiredfermentation pH 7.0 and temperature 30° C. An inoculum of E. coli straintaken from a frozen vial was streaked onto an LB broth agar plate (withantibiotics) and incubated at 37° C. A single colony was inoculated intotryptone-yeast extract medium. After the inoculum grew to OD 1.0,measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time, the glucose feed was decreased tomeet metabolic demands. The total amount of glucose delivered to thebioreactor during the 57 hour fermentation was 3.9 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 100 uMwhen the carbon dioxide evolution rate reached 100 mmol/L/hr. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 111A. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 1.6 g/L (FIG. 111B). The specificproductivity of isoprene over the course of the fermentation is shown inFIG. 111C and peaked at 1.2 mg/OD/hr. The total amount of isopreneproduced during the 57 hour fermentation was 16.2 g. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 0.9%. The weight percent yield of isoprene from glucose was 0.4%.

XIV. Production of Isoprene from E. coli BL21 Containing the KudzuIsoprene Synthase Using Glycerol as a Carbon Source

A 15-L scale fermentation of E. coli expressing Kudzu isoprene synthasewas used to produce isoprene from cells fed glycerol in fed-batchculture. This experiment demonstrates that growing cells in the presenceof glycerol (without glucose) resulted in the production of 2.2 mg/L ofisoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, and 1000× modified tracemetal solution 1 ml. All of the components were added together anddissolved in diH₂O. This solution was autoclaved. The pH was adjusted to7.0 with ammonium hydroxide (30%) and q.s. to volume. Glycerol 5.1 g,thiamine*HCl 0.1 g, and antibiotics were added after sterilization andpH adjustment.

1000× Modified Trace Metal Solution:

The medium was generated using the following components per literfermentation medium: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pTrcKudzu plasmid. This experiment was carried outto monitor isoprene formation from glycerol at the desired fermentationpH 7.0 and temperature 35° C. An inoculum of E. coli strain taken from afrozen vial was streaked onto an LA broth agar plate (with antibiotics)and incubated at 37° C. A single colony was inoculated intosoytone-yeast extract-glucose medium and grown at 35° C. After theinoculum grew to OD 1.0, measured at 550 nm, 600 mL was used toinoculate a 7.5-L bioreactor.

Glycerol was fed at an exponential rate until cells reached an opticaldensity at 550 nm (OD₅₅₀) of 153. The total amount of glycerol deliveredto the bioreactor during the 36 hour fermentation was 1.7 kg. Other thanthe glucose in the inoculum, no glucose was added to the bioreactor.Induction was achieved by adding IPTG. The IPTG concentration wasbrought to 20 uM when the OD₅₅₀ reached a value of 50. The OD₅₅₀ profilewithin the bioreactor over time is shown in FIG. 57. The isoprene levelin the off gas from the bioreactor was determined as described herein.The isoprene titer increased over the course of the fermentation to afinal value of 2.2 mg/L (FIG. 58). The total amount of isoprene producedduring the 54 hour fermentation was 20.9 mg, and the time course ofproduction is shown in FIG. 59.

XV. Isoprene Fermentation from E. coli Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L ScaleUsing Invert Sugar as a Carbon Source

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides and Kudzu isoprene synthase was used to produce isoprenefrom cells fed invert sugar in fed-batch culture. This experimentdemonstrates that growing cells in the presence of invert sugar resultedin the production of 2.4 g/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× Modified Trace Metal Solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Invert sugar 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. Thisexperiment was carried out to monitor isoprene formation from invertsugar at the desired fermentation pH 7.0 and temperature 30° C. Aninoculum of E. coli strain taken from a frozen vial was streaked onto anLB broth agar plate (with antibiotics) and incubated at 37° C. A singlecolony was inoculated into tryptone-yeast extract medium. After theinoculum grew to OD 1.0, measured at 550 nm, 500 mL was used toinoculate a 5-L bioreactor.

Invert sugar was fed at an exponential rate until cells reached thestationary phase. After this time the invert sugar feed was decreased tomeet metabolic demands. The total amount of invert sugar delivered tothe bioreactor during the 44 hour fermentation was 2.4 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 uMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 9. TheIPTG concentration was raised to 50 uM when OD₅₅₀ reached 200. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 96. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 2.4 g/L (FIG. 97). The total amount ofisoprene produced during the 44 hour fermentation was 18.4 g and thetime course of production is shown in FIG. 98. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 1.7%. The weight percent yield of isoprene from glucose was 0.8%.

Example 9 Construction of the Upper and Lower MVA Pathway forIntegration into Bacillus subtilis

I. Construction of the Upper MVA Pathway in Bacillus subtilis

The upper pathway from Enterococcus faecalis is integrated into B.subtilis under control of the aprE promoter. The upper pathway consistsof two genes; mvaE, which encodes for AACT and HMGR, and mvaS, whichencodes for HMGS. The two genes are fused together with a stop codon inbetween, an RBS site in front of mvaS, and are under the control of theaprE promoter. A terminator is situated after the mvaE gene. Thechloramphenicol resistance marker is cloned after the mvaE gene and theconstruct is integrated at the aprE locus by double cross over usingflanking regions of homology.

Four DNA fragments are amplified by PCR such that they contain overhangsthat will allowed them to be fused together by a PCR reaction. PCRamplifications are carried out using Herculase polymerase according tomanufacturer's instructions.

1. PaprE CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-94 (−) Fuse PaprE to mvaE(SEQ ID NO: 83) 5′-CAATAATAACTACTGTTTTCACTCTTTACCCTCTCCTTTTAATemplate: Bacillus subtilis chromosomal DNA 2. mvaECF 07-93 (+) fuse mvaE to the aprE promoter (GTG start codon)(SEQ ID NO: 84) 5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTGCF 07-62 (−) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 35)5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTTTCTTAAATCTemplate: Enterococcus faecalis chromosomal DNA (from ATCC) 3. mvaSCF 07-61 (+) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 36)5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGGATTGATAAACF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 85)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate: Enterococcus faecalis chromosomal DNA4. B. amyliquefaciens alkaline serine protease terminatorCF 07-123 (+) Fuse the end of mvaS to the terminator (SEQ ID NO: 102)5′-ACCGTTCGTTCTTATCGAAACTAAAAAAAACCGGCCTTGGCCCCGCF 07-46 (−) End of B. amyliquefaciens terminator BamHI (SEQ ID NO: 63)5′-GACATGACGGATCCGATTACGAATGCCGTCTCTemplate: Bacillus amyliquefaciens chromosomal DNA PCR Fusion Reactions5. Fuse mvaE to mvaSCF 07-93 (+) fuse mvaE to the aprE promoter (GTG start codon)(SEQ ID NO: 84) 5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTGCF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 85)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate: #2 and 3 from above 6. Fuse mvaE-mvaS to aprE promoterCF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGCCF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 85)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate #1 and #4 from above 7. Fuse PaprE-mvaE-mvaS to terminatorCF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGCCF 07-46 (−) End of B. amyliquefaciens terminator BamHI (SEQ ID NO: 63)5′-GACATGACGGATCCGATTACGAATGCCGTCTC Template: #4 and #6

The product is digested with restriction endonucleases PstI/BamHI andligated to pJM102 (Perego, M. 1993. Integrational vectors for geneticmanipulation in Bacillus subtilis, p. 615-624. In A. L. Sonenshein, J.A. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positivebacteria: biochemistry, physiology, and molecular genetics. AmericanSociety for Microbiology, Washington,D.C.) which is digested withPstI/BamHI. The ligation is transformed into E. coli TOP 10 chemicallycompetent cells and transformants are selected on LA containingcarbenicillin (50 μg/ml). The correct plasmid is identified bysequencing and is designated pJMUpperpathway2 (FIGS. 50 and 51).Purified plasmid DNA is transformed into Bacillus subtilis aprEnprEPxyl-comK and transformants are selected on L agar containingchloramphenicol (5 μg/ml). A correct colony is selected and is platedsequentially on L agar containing chloramphenicol 10, 15 and 25 μg/ml toamplify the number of copies of the cassette containing the upperpathway.

The resulting strain is tested for mevalonic acid production by growingin LB containing 1% glucose and 1%. Cultures are analyzed by GC for theproduction of mevalonic acid.

This strain is used subsequently as a host for the integration of thelower mevalonic acid pathway.

The following primers are used to sequence the various constructs above.

Sequencing primers: CF 07-134 (+) Start of aprE promoter PstI(SEQ ID NO: 82) 5′-GACATCTGCAGCTCCATTTTCTTCTGCCF 07-58 (+) Start of mvaE gene (SEQ ID NO: 38)5′-ATGAAAACAGTAGTTATTATTGATGC CF 07-59 (−) End of mvaE gene(SEQ ID NO: 39) 5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGCCF 07-82 (+) Start of mvaS gene (SEQ ID NO: 40)5′-ATGACAATTGGGATTGATAAAATTAG CF 07-83 (−) End of mvaS gene(SEQ ID NO: 41) 5′-TTAGTTTCGATAAGAACGAACGGTCF 07-86 (+) Sequence in mvaE (SEQ ID NO: 42) 5′-GAAATAGCCCCATTAGAAGTATCCF 07-87 (+) Sequence in mvaE (SEQ ID NO: 43)5′-TTGCCAATCATATGATTGAAAATC CF 07-88 (+) Sequence in mvaE(SEQ ID NO: 44) 5′-GCTATGCTTCATTAGATCCTTATCG CF 07-89 (+) Sequence mvaS(SEQ ID NO: 45) 5′-GAAACCTACATCCAATCTTTTGCCC

Transformants are selected on LA containing chloramphenicol at aconcentration of 5 μg/ml. One colony is confirmed to have the correctintegration by sequencing and is plated on LA containing increasingconcentrations of chloramphenicol over several days, to a final level of25 μg/ml. This results in amplification of the cassette containing thegenes of interest. The resulting strain is designated CF 455:pJMupperpathway#1×Bacillus subtilis aprEnprE Pxyl comK (amplified togrow on LA containing chloramphenicol 25 μg/ml).

II. Construction of the Lower MVA Pathway in Bacillus subtilis

The lower MVA pathway, consisting of the genes mvkl, pmk, mpd and idiare combined in a cassette consisting of flanking DNA regions from thenprE region of the B. subtilis chromosome (site of integration), theaprE promoter, and the spectinomycin resistance marker (see FIGS. 28 and29). This cassette is synthesized by DNA2.0 and is integrated into thechromosome of B. subtilis containing the upper MVA pathway integrated atthe aprE locus. The kudzu isoprene synthase gene is expressed from thereplicating plasmid described in Example 4 and is transformed into thestrain with both upper and lower pathways integrated.

Example 10 Exemplary Isoprene Compositions and Methods of Making them

Compositional analysis of fermentation off-gas containing isoprene

A 14 L scale fermentation was performed with a recombinant E. coli BL21(DE3) strain containing two plasmids (pCL upperMev; pTrcKKDyIkISencoding the full mevalonate pathway for isoprenoid precursorbiosynthesis, an isoprenyl pyrophosphate isomerase from yeast, and anisoprene synthase from Kudzu. Fermentation off-gas from the 14 L tankwas collected into 20 mL headspace vials at around the time of peakisoprene productivity (27.9 hours elapsed fermentation time, “EFT”) andanalyzed by headspace GC/MS for volatile components.

Headspace analysis was performed with an Agilent 6890 GC/MS systemfitted with an Agilent HP-5MS GC/MS column (30 m×250 μm; 0.25 μm filmthickness). A combiPAL autoinjector was used for sampling 500 uLaliquots from 20 mL headspace vials. The GC/MS method utilized helium asthe carrier gas at a flow of 1 mL/min. The injection port was held at250° C. with a split ratio of 50:1. The oven temperature was held at 37°C. for an initial 2 minute period, followed an increase to 237° C. at arate of 25° C./min for a total method time of 10 minutes. The Agilent5793N mass selective detector scanned from m/z 29 to m/z 300. The limitof detection of this system is approximately 0.1 ug/L_(gas) orapproximately 0.1 ppm. If desired, more sensitive equipment with a lowerlimit of detection may be used.

The off-gas consisted of 99.925% (v/v) permanent gases (N₂, CO₂ and O₂),approximately 0.075% isoprene (2-methyl-1,3-butadiene) (˜750 ppmv, 2100μg/L) and minor amounts (<50 ppmv) of ethanol, acetone, and two C5prenyl alcohols. The amount of water vapor was not determined but wasestimated to be equal to the equilibrium vapor pressure at 0° C. Thecomposition of the volatile organic fraction was determined byintegration of the area under the peaks in the GC/MS chromatogram (FIGS.86A and 86B) and is listed in Table 6. Calibration curves for ethanoland acetone standards enabled the conversion of GC area to gas phaseconcentration in units of ug/L using standard methods.

TABLE 6 Composition of volatile organic components in fermentationoff-gas. The off-gas was analyzed at the 27.9 hour time point of afermentation using an E. coli BL21 (DE3) strain expressing aheterologous mevalonate pathway, an isoprenyl pyrophosphate isomerasefrom yeast, and an isoprene synthase from Kudzu. Compound RT (min) GCarea Area % Conc. (ug/L) Ethanol 1.669 239005 0.84 62 +/− 6 Acetone1.703 288352 1.02 42 +/− 4 Isoprene (2-methyl- 1.829 27764544 97.81 2000+/− 200 1,3-butadiene) 3-methyl-3-buten-1-ol 3.493 35060 0.12 <103-methyl-2-buten-1-ol 4.116 58153 0.20 <10II. Measurement of Trace Volatile Organic Compounds (VOCs) Co-Producedwith Isoprene During Fermentation of a Recombinant E. coli Strain

A 14 L scale fermentation was performed with a recombinant E. coli BL21(DE3) strain containing two plasmids (pCL upperMev; pTrcKKDyIkIS)encoding the full mevalonate pathway for isoprenoid precursorbiosynthesis, an isoprenyl pyrophosphate isomerase from yeast, and anisoprene synthase from Kudzu.

Fermentation off-gas was passed through cooled headspace vials in orderto concentrate and identify trace volatile organic components. Theoff-gas from this fermentation was sampled at a rate of 1 L/min for 10minutes through a 20 mL headspace vial packed with quartz wool (2 g) andcooled to −78° C. with dry ice. The vial was recapped with a fresh vialcap and analyzed by headspace GC/MS for trapped VOCs using theconditions described in Example 10, part I. The ratios of compoundsobserved in FIGS. 87A-87D are a combination of overall level in thefermentation off-gas, the relative vapor pressure at −78° C., and thedetector response of the mass spectrometer. For example, the low levelof isoprene relative to oxygenated volatiles (e.g., acetone and ethanol)is a function of the high volatility of this material such that it doesnot accumulate in the headspace vial at −78° C.

The presence of many of these compounds is unique to isoprenecompositions derived from biological sources. The results are depictedin FIGS. 87A-87D and summarized in Tables 7A and 7B.

TABLE 7A Trace volatiles present in off-gas produced by E. coli BL21(DE3) (pCL upperMev; pTrcKKDyIkIS) following cryo-trapping at −78° C.Compound RT (min) GC Area¹ Area %² Ratio %³ Acetaldehyde 1.542 40198614.841 40.14 Ethanol 1.634 10553620 12.708 105.39 Acetone 1.727 72363238.714 72.26 2-methyl-1,3-butadiene 1.777 10013714 12.058 100.001-propanol 1.987 163574 0.197 1.63 Diacetyl 2.156 221078 0.266 2.212-methyl-3-buten-2-ol 2.316 902735 1.087 9.01 2-methyl-1-propanol 2.451446387 0.538 4.46 3-methyl-1-butanal 2.7 165162 0.199 1.65 1-butanol2.791 231738 0.279 2.31 3-methyl-3-buten-1-ol 3.514 14851860 17.884148.32 3-methyl-1-butanol 3.557 8458483 10.185 84.473-methyl-2-buten-1-ol 4.042 18201341 21.917 181.76 3-methyl-2-butenal4.153 1837273 2.212 18.35 3-methylbutyl acetate 5.197 196136 0.236 1.963-methyl-3-but-1-enyl acetate 5.284 652132 0.785 6.51 2-heptanone 5.34867224 0.081 0.67 2,5-dimethylpyrazine 5.591 58029 0.070 0.583-methyl-2-but-1-enyl acetate 5.676 1686507 2.031 16.846-methyl-5-hepten-2-one 6.307 101797 0.123 1.02 2,4,5-trimethylpyridine6.39 68477 0.082 0.68 2,3,5-trimethylpyrazine 6.485 30420 0.037 0.30(E)-3,7-dimethyl-1,3,6-octatriene 6.766 848928 1.022 8.48(Z)-3,7-dimethyl-1,3,6-octatriene 6.864 448810 0.540 4.483-methyl-2-but-1-enyl butyrate 7.294 105356 0.127 1.05 Citronellal 7.756208092 0.251 2.08 2,3-cycloheptenolpyridine 8.98 1119947 1.349 11.18 ¹GCarea is the uncorrected area under the peak corresponding to the listedcompound. ²Area % is the peak area expressed as a % relative to thetotal peak area of all compounds. ³Ratio % is the peak area expressed asa % relative to the peak area of 2-methyl-1,3-butadiene.

TABLE 7B Trace volatiles present in off-gas produced by E. coli BL21(DE3) (pCL upperMev; pTrcKKDyIkIS) following cryo-trapping at −196° C.Compound RT (min) GC Area¹ Area %² Ratio %³ Acetaldehyde 1.54 16557100.276 0.33 Methanethiol 1.584 173620 0.029 0.03 Ethanol 1.631 102596801.707 2.03 Acetone 1.722 73089100 12.164 14.43 2-methyl-1,3-butadiene1.771 506349429 84.269 100.00 methyl acetate 1.852 320112 0.053 0.061-propanol 1.983 156752 0.026 0.03 Diacetyl 2.148 67635 0.011 0.012-butanone 2.216 254364 0.042 0.05 2-methyl-3-buten-2-ol 2.312 6847080.114 0.14 ethyl acetate 2.345 2226391 0.371 0.44 2-methyl-1-propanol2.451 187719 0.031 0.04 3-methyl-1-butanal 2.696 115723 0.019 0.023-methyl-2-butanone 2.751 116861 0.019 0.02 1-butanol 2.792 54555 0.0090.01 2-pentanone 3.034 66520 0.011 0.01 3-methyl-3-buten-1-ol 3.5161123520 0.187 0.22 3-methyl-1-butanol 3.561 572836 0.095 0.11 ethylisobutyrate 3.861 142056 0.024 0.03 3-methyl-2-buten-1-ol 4.048 3025580.050 0.06 3-methyl-2-butenal 4.152 585690 0.097 0.12 butyl acetate4.502 29665 0.005 0.01 3-methylbutyl acetate 5.194 271797 0.045 0.053-methyl-3-but-1-enyl acetate 5.281 705366 0.117 0.143-methyl-2-but-1-enyl acetate 5.675 815186 0.136 0.16(E)-3,7-dimethyl-1,3,6-octatriene 6.766 207061 0.034 0.04(Z)-3,7-dimethyl-1,3,6-octatriene 6.863 94294 0.016 0.022,3-cycloheptenolpyridine 8.983 135104 0.022 0.03 ¹GC area is theuncorrected area under the peak corresponding to the listed compound.²Area % is the peak area expressed as a % relative to the total peakarea of all compounds. ³Ratio % is the peak area expressed as a %relative to the peak area of 2-methyl-1,3-butadiene.III. Absence of C5 Hydrocarbon Isomers in Isoprene Derived fromFermentation.

Cryo-trapping of isoprene present in fermentation off-gas was performedusing a 2 mL headspace vial cooled in liquid nitrogen. The off-gas (1L/min) was first passed through a 20 mL vial containing sodium hydroxidepellets in order to minimize the accumulation of ice and solid CO₂ inthe 2 mL vial (−196° C.). Approximately 10 L of off-gas was passedthrough the vial, after which it was allowed to warm to −78° C. withventing, followed by resealing with a fresh vial cap and analysis byGC/MS.

GC/MS headspace analysis was performed with an Agilent 6890 GC/MS systemusing a 100 uL gas tight syringe in headspace mode. A Zebron ZB-624GC/MS column (30 m×250 μm; 1.40 μm film thickness) was used forseparation of analytes. The GC autoinjector was fitted with a gas-tight100 uL syringe, and the needle height was adjusted to allow theinjection of a 50 uL headspace sample from a 2 mL GC vial. The GC/MSmethod utilized helium as the carrier gas at a flow of 1 mL/min. Theinjection port was held at 200° C. with a split ratio of 20:1. The oventemperature was held at 37° C. for the 5 minute duration of theanalysis. The Agilent 5793N mass selective detector was run in singleion monitoring (SIM) mode on m/z 55, 66, 67 and 70. Under theseconditions, isoprene was observed to elute at 2.966 minutes (FIG. 88B).A standard of petroleum derived isoprene (Sigma-Aldrich) was alsoanalyzed using this method and was found to contain additional C5hydrocarbon isomers, which eluted shortly before or after the main peakand were quantified based on corrected GC area (FIG. 88A).

TABLE 8A GC/MS analysis of petroleum-derived isoprene Area % of total C5Compound RT (min) GC area hydrocarbons 2-methyl-1-butene 2.689 18.2 ×10³ 0.017% (Z)-2-pentene 2.835 10.6 × 10⁴ 0.101% Isoprene 2.966 10.4 ×10⁷ 99.869% 1,3-cyclopentadiene 3.297 12.8 × 10³ 0.012% (CPD)

TABLE 8B GC/MS analysis of fermentation-derived isoprene (% total C5hydrocarbons) Corrected GC % of total C5 Compound RT (min) Areahydrocarbons Isoprene 2.966 8.1 × 10⁷ 100%

In a separate experiment, a standard mixture of C5 hydrocarbons wasanalyzed to determine if the detector response was the same for each ofthe compounds. The compounds were 2-methyl-1-butene,2-methyl-1,3-butadiene, (E)-2-pentene, (Z)-2-pentene and(E)-1,3-pentadiene. In this case, the analysis was performed on anAgilent DB-Petro column (100 m×0.25 mm, 0.50 um film thickness) held at50° C. for 15 minutes. The GC/MS method utilized helium as the carriergas at a flow of 1 mL/min. The injection port was held at 200° C. with asplit ratio of 50:1. The Agilent 5793N mass selective detector was runin full scan mode from m/z 19 to m/z 250. Under these conditions, a 100ug/L concentration of each standard produced the same detector responsewithin experimental error.

IV. Compositions Comprising Isoprene Adsorbed to a Solid Phase.

Biologically-produced isoprene was adsorped to activated carbonresulting in a solid phase containing 50 to 99.9% carbon, 0.1% to 50%isoprene, 0.01% to 5% water, and minor amounts (<0.1%) of other volatileorganic components.

Fermentation off-gas was run through a copper condensation coil held at0° C., followed by a granulated silica desiccant filter in order toremove water vapor. The dehumidified off-gas was then run through carboncontaining filters (Koby Jr, Koby Filters, MA) to the point at whichbreakthrough of isoprene was detected in the filter exhaust by GC/MS.The amount of isoprene adsorped to the cartridge can be determinedindirectly by calculating the concentration in the off-gas, the overallflow rate and the percent breakthrough over the collection period.Alternately the adsorped isoprene can be recovered from the filters bythermal, vacuum, or solvent-mediated desorption.

V. Collection and Analysis of Condensed Isoprene.

Fermentation off-gas is dehumidified, and the CO₂ removed by filtrationthrough a suitable adsorbant (e.g., ascarite). The resulting off-gasstream is then run through a liquid nitrogen-cooled condenser in orderto condense the VOCs in the stream. The collection vessel containst-butyl catechol to inhibit the resulting isoprene condensate. Thecondensate is analyzed by GC/MS and NMR in order to determine purityusing standard methods, such as those described herein.

VI. Production of Prenyl Alcohols by Fermentation

Analysis of off-gas from an E. coli BL21 (DE3) strain expressing a Kudzuisoprene synthase revealed the presence of both isoprene and3-methyl-3-buten-1-ol (isoprenol). The levels of the two compounds inthe fermentation off-gas over the fermentation are shown in FIG. 89 asdetermined by headspace GC/MS. Levels of isoprenol(3-methyl-3-buten-1-ol, 3-MBA) attained was nearly 10 ug/L_(offgas) inthis experiment. Additional experiments produced levels of approximately20 ug/L_(offgas) in the fermentation off-gas.

Example 11 The De-Coupling of Growth and Production of Isoprene in E.coli Expressing Genes from the Mevalonic Acid Pathway and Fermented in aFed-Batch Culture

Example 11 illustrates the de-coupling of cell growth from mevalonicacid and isoprene production.

I. Fermentation Conditions

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH2O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl2*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filtersterilized with a 0.22 micron filter.

Fermentation was performed with E. coli cells containing thepTrcHis2AUpperPathway (also called pTrcUpperMVA, FIGS. 91 and 92A-92C)(50 μg/ml carbenicillin) or the pCL PtrcUpperMVA (also called pCLPtrcUpperPathway (FIG. 26)) (50 μg/ml spectinomycin) plasmids. Forexperiments in which isoprene was produced, the E. coli cells alsocontained the pTrc KKDyIkIS (50 μg/mlkanamycin) plasmid. Theseexperiments were carried out to monitor mevalonic acid or isopreneformation from glucose at the desired fermentation pH 7.0 andtemperature 30° C. An inoculum of an E. coli strain taken from a frozenvial was streaked onto an LA broth agar plate (with antibiotics) andincubated at 37° C. A single colony was inoculated into tryptone-yeastextract medium. After the inoculum grew to optical density 1.0 whenmeasured at 550 nm, it was used to inoculate the bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. Induction was achieved by adding IPTG. The mevalonicacid concentration in fermentation broth was determined by applyingperchloric acid (Sigma-Aldrich #244252) treated samples (0.3 M incubatedat 4° C. for 5 minutes) to an organic acids HPLC column (BioRad#125-0140). The concentration was determined by comparing the brothmevalonic acid peak size to a calibration curve generated frommevalonolacetone (Sigma-Aldrich #M4667) treated with perchloric acid toform D,L-mevalonate. The isoprene level in the off gas from thebioreactor was determined as described herein. The isoprene titer isdefined as the amount of isoprene produced per liter of fermentationbroth.

II. Mevalonic Acid Production from E. coli BL21 (DE3) Cells Expressingthe pTrcUpperMVA Plasmid at a 150-L Scale

BL21 (DE3) cells that were grown on a plate as explained above inExample 11, part I were inoculated into a flask containing 45 mL oftryptone-yeast extract medium and incubated at 30° C. with shaking at170 rpm for 5 hours. This solution was transferred to a 5-L bioreactorof tryptone-yeast extract medium, and the cells were grown at 30° C. and27.5 rpm until the culture reached an OD₅₅₀ of 1.0. The 5 L of inoculumwas seeded into a 150-L bioreactor containing 45-kg of medium. The IPTGconcentration was brought to 1.1 mM when the OD₅₅₀ reached a value of10. The OD₅₅₀ profile within the bioreactor over time is shown in FIG.60A. The mevalonic acid titer increased over the course of thefermentation to a final value of 61.3 g/L (FIG. 60B). The specificproductivity profile throughout the fermentation is shown in FIG. 60Cand a comparison to FIG. 60A illustrates the de-coupling of growth andmevalonic acid production. The total amount of mevalonic acid producedduring the 52.5 hour fermentation was 4.0 kg from 14.1 kg of utilizedglucose. The molar yield of utilized carbon that went into producingmevalonic acid during fermentation was 34.2%.

III. Mevalonic Acid Production from E. coli BL21 (DE3) Cells Expressingthe pTrcUpperMVA Plasmid at a 15-L Scale

BL21 (DE3) cells that were grown on a plate as explained above inExample 11, part I were inoculated into a flask containing 500 mL oftryptone-yeast extract medium and grown at 30° C. at 160 rpm to OD₅₅₀1.0. This material was seeded into a 15-L bioreactor containing 4.5-kgof medium. The IPTG concentration was brought to 1.0 mM when the OD₅₅₀reached a value of 10. The OD₅₅₀ profile within the bioreactor over timeis shown in FIG. 61A. The mevalonic acid titer increased over the courseof the fermentation to a final value of 53.9 g/L (FIG. 61B). Thespecific productivity profile throughout the fermentation is shown inFIG. 61C and a comparison to FIG. 61A illustrates the de-coupling ofgrowth and mevalonic acid production. The total amount of mevalonic acidproduced during the 46.6 hour fermentation was 491 g from 2.1 kg ofutilized glucose. The molar yield of utilized carbon that went intoproducing mevalonic acid during fermentation was 28.8%.

IV. Mevalonic Acid Production from E. coli FM5 Cells Expressing thepTrcUpperMVA Plasmid at a 15-L Scale

FM5 cells that were grown on a plate as explained above in Example 11,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 1.0 mM when the OD₅₅₀ reached avalue of 30. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 62A. The mevalonic acid titer increased over the course of thefermentation to a final value of 23.7 g/L (FIG. 62B). The specificproductivity profile throughout the fermentation is shown in FIG. 62Cand a comparison to FIG. 62A illustrates the de-coupling of growth andmevalonic acid production. The total amount of mevalonic acid producedduring the 51.2 hour fermentation was 140 g from 1.1 kg of utilizedglucose. The molar yield of utilized carbon that went into producingmevalonic acid during fermentation was 15.2%.

V. Isoprene Production from E. coli BL21 (DE3) Cells Expressing the pCLPtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

BL21 (DE3) cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkISplasmids that were grown on a plate as explained above in Example 11,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 25 μM when the OD₅₅₀ reached avalue of 10. The IPTG concentration was raised to 50 uM when OD₅₅₀reached 190. The IPTG concentration was raised to 100 uM at 38 hours offermentation. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 63A. The isoprene titer increased over the course of thefermentation to a final value of 2.2 g/L broth (FIG. 63B). The specificproductivity profile throughout the fermentation is shown in FIG. 63Cand a comparison to FIG. 63A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the54.4 hour fermentation was 15.9 g from 2.3 kg of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 1.53%.

VI. Isoprene Production from E. coli BL21 (DE3) Tuner Cells Expressingthe pCL PtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

BL21 (DE3) tuner cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkISplasmids that were grown on a plate as explained above in Example 11,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 26 μM when the OD₅₅₀ reached avalue of 10. The IPTG concentration was raised to 50 uM when OD₅₅₀reached 175. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 64A. The isoprene titer increased over the course of thefermentation to a final value of 1.3 g/L broth (FIG. 64B). The specificproductivity profile throughout the fermentation is shown in FIG. 64Cand a comparison to FIG. 64A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the48.6 hour fermentation was 9.9 g from 1.6 kg of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 1.34%.

VII. Isoprene Production from E. coli MG1655 Cells Expressing the pCLPtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

MG1655 cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmidsthat were grown on a plate as explained above in Example 11, part I wereinoculated into a flask containing 500 mL of tryptone-yeast extractmedium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material wasseeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTGconcentration was brought to 24 μM when the OD₅₅₀ reached a value of 45.The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 65A.The isoprene titer increased over the course of the fermentation to afinal value of 393 mg/L broth (FIG. 65B). The specific productivityprofile throughout the fermentation is shown in FIG. 65C and acomparison to FIG. 65A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the67.4 hour fermentation was 2.2 g from 520 g of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 0.92%.

VIII. Isoprene Production from E. coli MG1655Ack-Pta Cells Expressingthe pCL PtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

MG1655ack-pta cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkISplasmids that were grown on a plate as explained above in Example 11,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 30 μM when the OD₅₅₀ reached avalue of 10. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 66A. The isoprene titer increased over the course of thefermentation to a final value of 368 mg/L broth (FIG. 66B). The specificproductivity profile throughout the fermentation is shown in FIG. 66Cand a comparison to FIG. 66A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the56.7 hour fermentation was 1.8 g from 531 g of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 0.73%.

IX. Isoprene Production from E. coli FM5 Cells Expressing the pCLPtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

FM5 cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmidsthat were grown on a plate as explained above in Example 11, part I wereinoculated into a flask containing 500 mL of tryptone-yeast extractmedium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material wasseeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTGconcentration was brought to 27 μM when the OD₅₅₀ reached a value of 15.The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 67A.The isoprene titer increased over the course of the fermentation to afinal value of 235 mg/L broth (FIG. 67B). The specific productivityprofile throughout the fermentation is shown in FIG. 67C and acomparison to FIG. 67A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the52.3 hour fermentation was 1.4 g from 948 g of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 0.32%.

Example 12 Production of Isoprene During the Exponential Growth Phase ofE. coli Expressing Genes from the Mevalonic Acid Pathway and Fermentedin a Fed-Batch Culture

Example 12 illustrates the production of isoprene during the exponentialgrowth phase of cells.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl2*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with ATCC11303 E. colicells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-Lbioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 50 hour fermentation was 2.0 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 uMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 10. TheIPTG concentration was raised to 50 uM when OD₅₅₀ reached 190. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 99. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 1.4 g/L (FIG. 100). The total amount ofisoprene produced during the 50 hour fermentation was 10.0 g. Theprofile of the isoprene specific productivity over time within thebioreactor is shown in FIG. 101. The molar yield of utilized carbon thatcontributed to producing isoprene during fermentation was 1.1%. Theweight percent yield of isoprene from glucose was 0.5%.

Example 13 Flammability Modeling and Testing of Isoprene I. Summary ofFlammability Modeling and Testing of Isoprene

Flammability modeling and experiments were performed for varioushydrocarbon/oxygen/nitrogen/water/carbon dioxide mixtures. This modelingand experimental tested was aimed at defining isoprene andoxygen/nitrogen flammability curves under specified steam and carbonmonoxide concentrations at a fixed pressure and temperature. A matrix ofthe model conditions is shown in Table 4, and a matrix of theexperiments performed is shown in Table 5.

TABLE 4 Summary of Modeled Isoprene Flammability Carbon Tem- SteamDioxide Isoprene Oxygen per- Pres- Concen- Concen- Concen- Concen- aturesure tration tration tration tration Series (° C.) (psig) (wt %) (wt. %)(vol. %) (vol. %) A 40 0 0  0 Varying Varying B 40 0 4  0 VaryingVarying C 40 0 0  5 Varying Varying D 40 0 0 10 Varying Varying E 40 0 015 Varying Varying F 40 0 0 20 Varying Varying G 40 0 0 30 VaryingVarying

TABLE 5 Summary of Isoprene Flammability Tests Isoprene Oxygen SteamConcen- Concen- Series Temperature Pressure Concentration trationtration Number (° C.) (psig) (vol. %) (vol. %) (vol. %) 1 40 0 0 VaryingVarying 2 40 0 4 Varying Varying

II. Description of Calculated Adiabatic Flame Temperature (CAFT) Model

Calculated adiabatic flame temperatures (CAFT) along with a selectedlimit flame temperature for combustion propagation were used todetermine the flammability envelope for isoprene. The computer programused in this study to calculate the flame temperatures is the NASA GlennResearch Center CEA (Chemical Equilibrium with Applications) software.

There are five steps involved in determining the flammability envelopeusing an adiabatic flame temperature model for a homogeneous combustionmechanism (where both the fuel and oxidant are in the gaseous state):selection of the desired reactants, selection of the test condition,selection of the limit flame temperature, modification of the reactants,and construction of a flammability envelope from calculations.

In this first step, selection of desired reactants, a decision must bemade as to the reactant species that will be present in the system andthe quantities of each. In many cases the computer programs used for thecalculations have a list of reactant and product species. If any of thedata for the species to be studied are not found in the program, theymay be obtained from other sources such as the JANAF tables or from theinternet. In this current model data for water, nitrogen, oxygen andcarbon dioxide were present in the program database. The programdatabase did not have isoprene as a species; therefore the thermodynamicproperties were incorporated manually.

The next step is to decide whether the initial pressure and temperatureconditions that the combustion process is taking place in. In this modelthe pressure was 1 atmosphere (absolute) and the temperature was 40° C.,the boiling point of isoprene.

The limit flame temperature for combustion can be either selected basedon theoretical principles or determined experimentally. Each method hasits own limitations.

Based on prior studies, the limit flame temperatures of hydrocarbonsfall in the range of 1000 K to 1500 K. For this model, the value of 1500K was selected. This is the temperature at which the reaction of carbonmonoxide to carbon dioxide (a highly exothermic reaction and constitutesa significant proportion of the flame energy) becomes self sustaining.

Once the limit flame temperature has been decided upon, modelcalculations are performed on the given reactant mixture (speciesconcentrations) and the adiabatic flame temperature is determined. Flamepropagation is considered to have occurred only if the temperature isgreater than the limit flame temperature. The reactant mixturecomposition is then modified to create data sets for propagation andnon-propagation mixtures.

This type of model shows good agreement with the experimentallydetermined flammability limits. Regions outside the derived envelope arenonflammable and regions within it are flammable. The shape of theenvelope forms a nose. The nose of the envelope is related to thelimiting oxygen concentration (LOC) for gaseous fuels.

III. Results from Calculated Adiabatic Flame Temperature (CAFT) Model

Plotted in FIGS. 68 through 74 are the CAFT model results for Series Ato G, respectively. The figures plot the calculated adiabatic flametemperature (using the NASA CEA program) as a function of fuelconcentration (by weight) for several oxygen/nitrogen ratios (byweight). The parts of the curve that are above 1500 K, the selectedlimit flame temperature, contain fuel levels sufficient for flamepropagation. The results may be difficult to interpret in the formpresented in FIGS. 68 through 74. Additionally, the current form is notconducive to comparison with experimental data which is generallypresented in terms of volume percent.

Using Series A as an example the data in FIG. 68 can be plotted in theform of a traditional flammability envelope. Using FIG. 68 and readingacross the 1500 K temperature line on the ordinate one can determine thefuel concentration for this limit flame temperature by dropping atangent to the abscissa for each curve (oxygen to nitrogen ratio) thatit intersects. These values can then be tabulated as weight percent offuel for a given weight percent of oxidizer (FIG. 75A). Then knowing thecomposition of the fuel (100 wt. % isoprene) and the composition of theoxidizer (relative content of water, oxygen and nitrogen) molarquantities can be established.

From these molar quantities percentage volume concentrations can becalculated. The concentrations in terms of volume percent can then beplotted to generate a flammability envelope (FIG. 75B). The area boundedby the envelope is the explosible range and the area excluded is thenon-explosible range. The “nose” of the envelope is the limiting oxygenconcentration. FIGS. 76A and 76B contain the calculated volumeconcentrations for the flammability envelope for Series B generated fromdata presented in FIG. 69. A similar approach can be used on datapresented in FIGS. 70-74.

IV. Flammability Testing Experimental Equipment and Procedure

Flammability testing was conducted in a 4 liter high pressure vessel.The vessel was cylindrical in shape with an inner diameter of 6″ and aninternal height of 8.625″. The temperature of the vessel (and the gasesinside) was maintained using external heaters that were controlled by aPID controller. To prevent heat losses, ceramic wool and reflectiveinsulation were wrapped around the pressure vessel. Type K thermocoupleswere used the measure the temperature of the gas space as well as thetemperature of the vessel itself. FIG. 77 illustrates the test vessel.

Before a test was ran, the vessel was evacuated and purged with nitrogento ensure that any gases from previous tests were removed. A vacuum wasthen pulled on the vessel. The pressure after this had been done wastypically around 0.06 bar(a). Due to the nitrogen purging, the gasresponsible for this initial pressure was assumed to be nitrogen. Usingpartial pressures, water, isoprene, nitrogen, and oxygen were then addedin the appropriate amounts to achieve the test conditions in question. Amagnetically driven mixing fan within the vessel ensured mixing of thegaseous contents. The gases were allowed to mix for about 2 minutes withthe fan being turned off approximately 1 minute prior to ignition.

The igniter was comprised of a 1.5 ohm nicrome coil and an AC voltagesource on a timer circuit. Using an oscilloscope, it was determined that34.4 VAC were delivered to the igniter for 3.2 seconds. A maximumcurrent of 3.8 amps occurred approximately halfway into the ignitioncycle. Thus, the maximum power was 131 W and the total energy providedover the ignition cycle was approximately 210 J.

Deflagration data was acquired using a variable reluctance ValidyneDP215 pressure transducer connected to a data acquisition system. A gasmixture was considered to have deflagrated if the pressure rise wasgreater than or equal to 5%.

V. Results of Flammability Testing

The first experimental series (Series 1) was run at 40° C. and 0 psigwith no steam. Running tests at varying concentrations of isoprene andoxygen produced the flammability curve shown in FIG. 78A. The datapoints shown in this curve are only those that border the curve. Adetailed list of all the data points taken for this series is shown inFIGS. 80A and 80B.

FIG. 78B summarizes the explosibility data points shown in FIG. 78A.FIG. 78C is a comparison of the experimental data with the CAFT modelpredicted flammability envelope. The model agrees very well with theexperimental data. Discrepancies may be due to the non-adiabatic natureof the test chamber and limitations of the model. The model looks at aninfinite time horizon for the oxidation reaction and does not take intoconsideration any reaction kinetic limitation.

Additionally, the model is limited by the number of equilibrium chemicalspecies that are in its database and thus may not properly predictpyrolytic species. Also, the flammability envelope developed by themodel uses one value for a limit flame temperature (1500K). The limitflame temperature can be a range of values from 1,000K to 1,500Kdepending on the reacting chemical species. The complex nature ofpyrolytic chemical species formed at fuel concentrations above thestoichiometric fuel/oxidizer level is one reason why the model may notaccurately predict the upper flammable limit for this system.

The second experimental series (Series 2) was run at 40° C. and 0 psigwith a fixed steam concentration of 4%. Running tests at varyingconcentrations of isoprene and oxygen produced the flammability curveshown in FIG. 79A. The data points shown in this curve are only thosethat border the curve. A detailed list of all the data points taken forthis series is shown in FIG. 81. Due to the similarity between the datain Series 1 only the key points of lower flammable limit, limitingoxygen concentration, and upper flammable limits were tested. Theaddition of 4% steam to the test mixture did not significantly changethe key limits of the flammability envelope. It should be noted thathigher concentrations of steam/water and or other inertants mayinfluence the flammability envelope.

FIG. 79B summarizes the explosibility data points shown in FIG. 79A.FIG. 79C is a comparison of the experimental data with the CAFT modelpredicted flammability envelope. The model agrees very well with theexperimental data. Discrepancies may be due to the same factorsdescribed in Series 1

V. Calculation of Flammability Limits of Isoprene in Air at 3Atmospheres of Pressure

The methods described in Example 13, parts Ito IV were also used tocalculate the flammability limits of isoprene at an absolute systempressure of 3 atmospheres and 40° C. These results were compared tothose of Example 13, parts Ito IV at an absolute system pressure of 1atmosphere and 40° C. This higher pressure was tested because theflammability envelope expands or grows larger as the initial systempressure is increased. The upper flammability limit is affected themost, followed by the limiting oxygen composition. The lowerflammability limit is the least affected (see, for example, “Bulletin627-Flammability Characteristics of Combustible Gases and Vapors”written by Michael G. Zabetakis and published by the former US Bureau ofMines (1965), which is hereby incorporated by reference in its entirety,particular with respect to the calculation of flammability limits).

In FIG. 82, the calculated adiabatic flame temperature is plotted as afunction of isoprene (fuel) concentration, expressed in weight percentof the total fuel/nitrogen/oxygen, where the system pressure wasinitially 3 atmospheres. The calculated flame temperatures are verysimilar to those determined initially in the 1 atmosphere system (FIG.83). As a result, when flammability envelopes are generated using thecalculated adiabatic flammability data, the curves are very similar (seeFIGS. 84 and 85). Therefore, based on these theoretical calculations, asystem pressure increase from 1 atmosphere to 3 atmosphere does notresult in a significant increase/broadening of the flammabilityenvelope. If desired, these model results may be validated usingexperimental testing (such as the experimental testing described hereinat a pressure of 1 atmosphere).

VII. Summary of Flammability Studies

A calculated adiabatic temperature model was developed for theflammability envelope of the isoprene/oxygen/nitrogen/water/carbondioxide system at 40° C. and 0 psig. The CAFT model that was developedagreed well with the experimental data generated by the tests conductedin this work. The experimental results from Series 1 and 2 validated themodel results from Series A and B.

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of skill in theart to which this invention belongs. Singleton, et al., Dictionary ofMicrobiology and Molecular Biology, 2nd ed., John Wiley and Sons, NewYork (1994), and Hale & Marham, The Harper Collins Dictionary ofBiology, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. It is tobe understood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary. Oneof skill in the art will also appreciate that any methods and materialssimilar or equivalent to those described herein can also be used topractice or test the invention.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole.

For use herein, unless clearly indicated otherwise, use of the terms“a”, “an,” and the like refers to one or more.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

APPENDIX 1 Exemplary 1-deoxy-D-xylulose-5-phosphate Synthase NucleicAcids and Polypeptides ATH: AT3G21500(DXPS1) AT4G15560(CLA1)AT5G11380(DXPS3) OSA: 4338768 4340090 4342614 CME: CMF089C PFA:MAL13P1.186 TAN: TA20470 TPV: TP01_(—)0516

ECO: b0420(dxs)ECJ: JWO410(dxs)ECE: Z0523(dxs)

ECS: ECs0474

ECC: c0531(dxs)ECI: UTI89—C0443(dxs)

ECP: ECP_(—)0479

ECV: APECO1_(—)1590(dxs)ECW: EcE24377A_(—)0451(dxs)

ECX: EcHS_A0491

STY: STY0461(dxs)STT: t2441(dxs)SPT: SPA2301(dxs)SEC: SCO₄₆₃(dxs)STM: STM0422(dxs)YPE: YP03177(dxs)YPK: y1008(dxs)YPM: YP_(—)0754(dxs)

YPA: YPA 2671 YPN: YPN_(—)0911 YPP: YPDSF_(—)2812

YPS: YPTB0939(dxs)YPI: YpsIP31758_(—)3112(dxs)SFL: SF0357(dxs)SFX: S0365(dxs)SFV: SFV_(—)0385(dxs)SSN: SSON_(—)0397(dxs)SBO: SBO_(—)0314(dxs)SDY: SDY_(—)0310(dxs)ECA: ECA1131(dxs)PLU: p1u3887(dxs)BUC: BU464(dxs)BAS: BUsg448(dxs)WBR: WGLp144(dxs)

SGL: SG0656

KPN: KPN_(—)00372(dxs)BFL: Bfl238(dxs)BPN: BPEN_(—)244(dxs)HIN: HI1439(dxs)HIT: NTHI1691(dxs)

HIP: CGSHiEE_(—)04795 HIQ: CGSHiGG_(—)01080

HDU: HD0441(dxs)HSO: HS_(—)0905(dxs)PMU: PM0532(dxs)MSU: MS1059(dxs)APL: APL_(—)0207(dxs)

XFA: XF2249

XFT: PD1293(dxs)XCC: XCC2434(dxs)

XCB: XC_(—)1678

XCV: XCV2764(dxs)XAC: XAC2565(dxs)XOO: XOO2017(dxs)

XOM: XOO_(—)1900(XOO1900) VCH: VC0889 VVU: VV1_(—)0315 VVY: VV0868 VPA:VP0686 VFI: VF0711 PPR: PBPRA0805

PAE: PA4044(dxs)PAU: PA14_(—)11550(dxs)PAP: PSPA7_(—)1057(dxs)PPU: PP_(—)0527(dxs)PST: PSPTO_(—)0698(dxs)

PSB: Psyr_(—)0604

PSP: PSPPH_(—)0599(dxs)PFL: PFL_(—)5510(dxs)

PFO: Pfl_(—)5007

PEN: PSEEN0600(dxs)

PMY: Pmen_(—)3844

PAR: Psyc_(—)0221(dxs)

PCR: Pcryo_(—)0245

ACI: ACIAD3247(dxs)SON: SO_(—)1525(dxs)

SDN: Sden_(—)2571 SFR: Sfri_(—)2790 SAZ: Sama_(—)2436 SBL: Sbal_(—)1357SLO: Shew_(—)2771 SHE: Shewmr4_(—)2731 SHM: Shewmr7_(—)2804 SHN:Shewana3_(—)2901 SHW: Sputw3181_(—)2831

ILO: IL2138(dxs)CPS: CPS_(—)1088(dxs)PHA: PSHAa2366(dxs)

PAT: Pat1_(—)1319 SDE: Sde_(—)3381 PIN: Ping_(—)2240 MAQ: Maqu_(—)2438

MCA: MCA0817(dxs)FTU: FTT1018c(dxs)FTF: FTF1018c(dxs)FTW: FTW_(—)0925(dxs)

FTL: FTL_(—)1072

FTH: FTH_(—)1047(dxs)FTA: FTA_(—)1131(dxs)FTN: FTN_(—)0896(dxs)

NOC: Noc_(—)1743 AEH: Mlg_(—)1381

HCH: HCH_(—)05866(dxs)

CSA: Csal_(—)0099

ABO: ABO_(—)2166(dxs)AHA: AHA_(—)3321(dxs)BCI: BCl_(—)0275(dxs)

RMA: Rmag_(—)0386

VOK: COSY_(—)0360(dxs)

NME: NMB 1867

NMA: NMA0589(dxs)NMC: NMC0352(dxs)

NGO: NGO0036

CVI: CV_(—)2692(dxs)RSO: RSc2221(dxs)

REU: Reut_A0882

REH: H16—A2732(dxs)

RME: Rmet_(—)2615

BMA: BMAA0330(dxs)BMV: BMASAVP1_(—)1512(dxs)BML: BMA10299_(—)1706(dxs)BMN: BMA10247—A0364(dxs)

BXE: Bxe_B2827 BUR: Bcep18194_B2211 BCN: Bcen_(—)4486 BCH:Bcen2424_(—)3879 BAM: Bamb_(—)3250

BPS: BPSS 1762(dxs)BPM: BURPS1710b_A0842(dxs)BPL: BURPS1106A_A2392(dxs)BPD: BURPS668—A2534(dxs)BTE: BTH_II0614(dxs)BPE: BP2798(dxs)BPA: BPP2464(dxs)BBR: BB1912(dxs)

RFR: Rfer_(—)2875 POL: Bpro_(—)1747 PNA: Pnap_(—)1501 AJS: Ajs_(—)1038MPT: Mpe_A2631

HAR: HEAR0279(dxs)MMS: mma_(—)0331NEU: NE1161(dxs)

NET: Neut_(—)1501 NMU: Nmul_A0236

EBA: ebA4439(dxs)AZO: azo1198(dxs)

DAR: Daro_(—)3061 TBD: Tbd_(—)0879 MFA: Mfla_(—)2133

HPY: HP0354(dxs)HPJ: jhp0328(dxs)

HPA: HPAG1_(—)0349

HHE: HH0608(dxs)HAC: Hac_(—)0968(dxs)

WSU: WS1996 TDN: Tmden_(—)0475

CJE: Cj0321(dxs)CJR: CJE0366(dxs)CJJ: CJJ81176_(—)0343(dxs)CJU: C8J_(—)0298(dxs)CJD: JJD26997_(—)1642(dxs)CFF: CFF8240_(—)0264(dxs)CCV: CCV52592_(—)1671(dxs) CCV52592_(—)1722CHA: CHAB381_(—)1297(dxs)CCO: CCC13826_(—)1594(dxs)ABU: Abu_(—)2139(dxs)NIS: NIS_(—)0391(dxs)SUN: SUN_(—)2055(dxs)GSU: GSU0686(dxs-1) GSU1764(dxs-2)

GME: Gmet_(—)1934 Gmet_(—)2822 PCA: Pcar_(—)1667 PPD: Ppro_(—)1191Ppro_(—)2403

DVU: DVU1350(dxs)

DVL: Dvul_(—)1718 DDE: Dde_(—)2200

LIP: LI0408(dsx)

DPS: DP2700 ADE: Adeh_(—)1097

MXA: MXAN_(—)4643(dxs)

SAT: SYN_(—)02456 SFU: Sfum_(—)1418

PUB: SAR11_(—)0611(dxs)MLO: mlr7474

MES: Meso_(—)0735

SME: SMc00972(dxs)ATU: Atu0745(dxs)

ATC: AGR_C_(—)1351

RET: RHE_CH00913(dxs)RLE: RL0973(dxs)

BME: BMEI1498

BMF: BAB 1_(—)0462(dxs)BMS: BR0436(dxs)BMB: BruAbl_(—)0458(dxs)BOV: BOV_(—)0443(dxs)BJA: b112651(dxs)BRA: BRADO2161(dxs)BBT: BBta_(—)2479(dxs)RPA: RPA0952(dxs)

RPB: RPB_(—)4460 RPC: RPC_(—)1149 RPD: RPD_(—)4305 RPE: RPE_(—)1067 NWI:Nwi_(—)0633 NHA: Nham_(—)0778

BHE: BH04350(dxs)BQU: BQ03540(dxs)BBK: BARBAKC583_(—)0400(dxs)

CCR: CC_(—)2068

SIL: SPO0247(dxs)

SIT: TM1040_(—)2920

RSP: RSP_(—)0254(dxsA) RSP_(—)1134(dxs)

JAN: Jann_(—)0088 Jann_(—)0170

RDE: RD1_(—)0101(dxs) RD1_(—)0548(dxs)

MMR: Mmar10_(—)0849

HNE: HNE_(—)1838(dxs)ZMO: ZMO1234(dxs) ZMO1598(dxs)

NAR: Saro_(—)0161 SAL: Sala_(—)2354 ELI: ELI 12520 GOX: GOX0252 GBE:GbCGDNIH1_(—)0221 GbCGDNIH1_(—)2404 RRU: Rru_A0054 Rru_A2619

MAG: amb2904

MGM: Mmc1_(—)1048 SUS: Acid_(—)1783

BSU: BG11715(dxs)

BHA: BH2779

BAN: BA4400(dxs)BAR: GBAA4400(dxs)

BAA: BA_(—)4853 BAT: BAS4081

BCE: BC4176(dxs)BCA: BCE_(—)4249(dxs)BCZ: BCZK3930(dxs)BTK: BT9727_(—)3919(dxs)BTL: BALH_(—)3785(dxs)BLI: BL01523(dxs)BLD: BLi02598(dxs)BCL: ABC2462(dxs)

BAY: RBAM_(—)022600 BPU: BPUM_(—)2159 GKA: GK2392 GTN: GTNG_(—)2322

LMO: lmo1365(tktB)LMF: LMOf2365_(—)1382(dxs)LIN: linl402(tktB)LWE: lwel380(tktB)LLA: L108911(dxsA) L123365(dxsB)

LLC: LACR_(—)1572 LACR_(—)1843

LLM: 11 mg_(—)0749(dxsB)

SAK: SAK_(—)0263

LPL: lp_(—)2610(dxs)

LJO: LJ0406 LAC: LBA0356

LSL: LSL_(—)0209(dxs)

LGA: LGAS_(—)0350 STH: STH1842

CAC: CAC2077 CA_P0106(dxs)

CPE: CPE1819

CPF: CPF_(—)2073(dxs)CPR: CPR_(—)1787(dxs)

CTC: CTC01575 CNO: NT01CX_(—)1983 CTH: Cthe_(—)0828

CDF: CD1207(dxs)CBO: CBO1881(dxs)CBA: CLB_(—)1818(dxs)CBH: CLC_(—)1825(dxs)CBF: CLI_(—)1945(dxs)CKL: CKL_(—)1231(dxs)CHY: CHY_(—)1985(dxs)

DSY: DSY2348 DRM: Dred_(—)1078

PTH: PTH_(—)1196(dxs)

SWO: Swol_(—)0582 CSC: Csac_(—)1853

TTE: TTE1298(dxs)

MTA: Moth_(—)1511 MPE: MYPE730

MGA: MGA_(—)1268(dxs)MTU: Rv2682c(dxs1) Rv3379c(dxs2)MTC: MT2756(dxs)MBO: Mb2701c(dxs1) Mb3413c(dxs2)MLE: ML1038(dxs)MPA: MAP2803c(dxs)MAV: MAV_(—)3577(dxs)MSM: MSMEG_(—)2776(dxs)

MMC: Mmcs_(—)2208

CGL: NCg11827(cg11902)CGB: cg2083(dxs)

CEF: CE1796

CDI: DIP1397(dxs)CJK: jk1078(dxs)NFA: nfa37410(dxs)

RHA: RHA1_ro06843 SCO: SCO6013(SC1C3.01) SCO6768(SC6A5.17)

SMA: SAV1646(dxs1) SAV2244(dxs2)

TWH: TWT484 TWS: TW280(Dxs)

LXX: Lxx10450(dxs)CMI: CMM_(—)1660(dxsA)AAU: AAur_(—)1790(dxs)

PAC: PPA1062 TFU: Tfu_(—)1917 FRA: Francci3_(—)1326

FAL: FRAAL2088(dxs)

ACE: Acel_(—)1393

SEN: SACE_(—)1815(dxs) SACE_(—)4351BLO: BL1132(dxs)BAD: BAD_(—)0513(dxs)

FNU: FN₁₂O₈ FN1464

RBA: RB2143(dxs)CTR: CT331(dxs)CTA: CTA_(—)0359(dxs)

CMU: TC0608

CPN: CPn1060(tktB_(—)2)

CPA: CP0790

CPJ: CPj1060(tktB_(—)2)

CPT: CpB 1102

CCA: CCA00304(dxs)CAB: CAB301(dxs)CFE: CF0699(dxs)PCU: pc0619(dxs)

TPA: TP0824

TDE: TDE1910(dxs)LIL: LA3285(dxs)LIC: LIC10863(dxs)LBJ: LBJ_(—)0917(dxs)LBL: LBL_(—)0932(dxs)SYN: sll1945(dxs)

SYW: SYNW1292(Dxs)

SYC: sycl087_c(dxs)

SYF: Synpcc7942_(—)0430 SYD: Syncc9605_(—)1430 SYE: Syncc9902_(—)1069

SYG: sync_(—)1410(dxs)SYR: SynRCC307_(—)1390(dxs)SYX: SynWH7803_(—)1223(dxs)CYA: CYA_(—)1701(dxs)CYB: CYB_(—)1983(dxs)TEL: tll0623GVI: gll0194ANA: alr0599

AVA: Ava_(—)4532

PMA: Pro0928(dxs)

PMM: PMM0907(Dxs)

PMT: PMT0685(dxs)

PMN: PMN2A_(—)0300 PMI: PMT9312_(—)0893

PMB: A9601_(—)09541(dxs)PMC: P9515_(—)09901(dxs)PMF: P9303_(—)15371(dxs)PMG: P9301_(—)09521(dxs)

PMH: P9215_(—)09851 PMJ: P9211_(—)08521

PME: NATL1_(—)09721(dxs)

TER: Tery_(—)3042 BTH: BT_(—)1403 BT_(—)4099 BFR: BF0873 BF4306

BFS: BF0796(dxs) BF4114PGI: PG2217(dxs)CHU: CHU_(—)3643(dxs)GFO: GFO_(—)3470(dxs)FPS: FP0279(dxs)CTE: CT0337(dxs)

CPH: Cpha266_(—)0671 PVI: Cvib_(—)0498 PLT: Plut_(—)0450

DET: DET0745(dxs)DEH: cbdb_A720(dxs)

DRA: DR_(—)1475 DGE: Dgeo_(—)0994 TTH: TTC1614 TTJ: TTHA0006

AAE: aq_(—)881

TMA: TM1770 PMO: Pmob_(—)1001 Exemplary Acetyl-CoA-AcetyltransferaseNucleic Acids and Polypeptides HSA: 38(ACAT1) 39(ACAT2) PTR:451528(ACAT1) MCC: 707653(ACAT1) 708750(ACAT2) MMU: 110446(Acat1)110460(Acat2) RNO: 25014(Acat1) CFA: 484063(ACAT2) 489421(ACAT1)

GGA: 418968(ACAT1) 421587(RCJMB04_(—)34i5)

XLA: 379569(MGC69098) 414622(MGC81403) 414639(MGC81256)

-   -   444457(MGC83664)        XTR: 394562(acat2)        DRE: 30643(acat2)

SPU: 759502(LOC759502) DME: Dmel_CG10932 Dmel_CG9149

CEL: TO2G5.4 TO2G5.7 TO2G5.8(kat-1)

ATH: AT5G48230(ACAT2/EMB1276) OSA: 4326136 4346520 CME: CMA042C CME087CSCE: YPL028W(ERG10) AGO: AGOS_ADR165c PIC: PICST_(—)31707(ERG10)

CAL: Ca019.1591(erg10)

CGR: CAGLOL12364 g SPO: SPBC215.09c MGR: MGG_(—)01755 MGG_(—)13499 ANI:AN1409.2 AFM: AFUA_(—)6G14200 AFUA_(—)8G04000 AOR: AO090103000012AO090103000406 CNE: CNC05280 UMA: UM03571.1 DDI: DDB 0231621 PFA:PF14_(—)0484 TET: TTHERM_(—)00091590 TTHERM_(—)00277470TTHERM_(—)00926980 TCR: 511003.60

ECO: b2224(atoB)ECJ: JW2218(atoB) JW5453(yqeF)ECE: Z4164(yqeF)

ECS: ECs3701

ECC: c2767(atoB) c3441(yqeF)ECI: UTI89—C2506(atoB) UTI89—C3247(yqeF)

ECP: ECP_(—)2268 ECP_(—)2857

ECV: APECO1_(—)3662(yqeF) APECO1_(—)4335(atoB) APECO1_(—)43352(atoB)

ECX: EcHS_A2365

STY: STY3164(yqeF)STT: t2929(yqeF)SPT: SPA2886(yqeF)SEC: SC2958(yqeF)STM: STM3019(yqeF)SFL: SF2854(yqeF)SFX: 53052(yqeF)SFV: SFV_(—)2922(yqeF)SSN: SSON_(—)2283(atoB) SSON_(—)3004(yqeF)SBO: SBO_(—)2736(yqeF)ECA: ECAl282(atoB)

ENT: Ent638_(—)3299 SPE: Spro_(—)0592

HIT: NTHI0932(atoB)XCC: XCC1297(atoB)

XCB: XC_(—)2943

XCV: XCV1401(thlA)XAC: XAC1348(atoB)XOO: XOO1881(atoB)

XOM: XOO_(—)1778(XOO1778) VCH: VCA0690 VCO: VCO395_(—)0630 VVU:VV2_(—)0494 VV2_(—)0741 VVY: VVA1043 VVA1210 VPA: VPA0620 VPA1123VPAl204 PPR: PBPRB 1112 PBPRB 1840

PAE: PA2001(atoB) PA2553 PA3454 PA3589 PA3925PAU: PA14_(—)38630(atoB)PPU: PP_(—)2051(atoB) PP_(—)2215(fadAx) PP_(—)3754 PP_(—)4636

PPF: Pput_(—)2009 Pput_(—)2403 Pput_(—)3523 Pput_(—)4498

PST: PSPTO_(—)0957(phbA-1) PSPTO_(—)3164(phbA-2)

PSB: Psyr_(—)0824 Psyr_(—)3031

PSP: PSPPH_(—)0850(phbA1) PSPPH_(—)2209(phbA2)PFL: PFL_(—)1478(atoB-2) PFL_(—)2321 PFL_(—)3066 PFL_(—)4330(atoB-2)PFL_(—)5283

PFO: Pfl_(—)1269 Pfl_(—)1739 Pfl_(—)2074 Pfl_(—)2868

PEN: PSEEN3197 PSEEN3547(fadAx) PSEEN4635(phbA)

PMY: Pmen_(—)1138 Pmen_(—)2036 Pmen_(—)3597 Pmen_(—)3662 Pmen_(—)3820PAR: Psyc_(—)0252 Psyc_(—)1169 PCR: Pcryo_(—)0278 Pcryo_(—)1236Pcryo_(—)1260 PRW: PsycPRwf 2011

ACI: ACIAD0694 ACIAD1612 ACIAD2516(atoB)SON: SO_(—)1677(atoB)

SDN: Sden_(—)1943 SFR: Sfri_(—)1338 Sfri_(—)2063 SAZ: Sama_(—)1375 SBL:Sbal_(—)1495 SBM: Shew185_(—)1489 SBN: Sba1195_(—)1525 SLO: Shew_(—)1667Shew_(—)2858 SPC: Sputcn32_(—)1397 SSE: Ssed_(—)1473 Ssed_(—)3533 SPL:Spea_(—)2783 SHE: Shewmr4_(—)2597 SHM: Shewmr7_(—)2664 SHN:Shewana3_(—)2771 SHW: Sputw3181_(—)2704 ILO: IL0872 CPS: CPS_(—)1605CPS_(—)2626

PHA: PSHAa0908 PSHAa1454(atoB) PSHAa1586(atoB)

PAT: Pat1_(—)2923 SDE: Sde_(—)3149 PIN: Ping_(—)0659 Ping_(—)2401 MAQ:Maqu_(—)2117 Maqu_(—)2489 Maqu_(—)2696 Maqu_(—)3162 CBU: CBU_(—)0974

LPN: lpg1825(atoB)LPF: lpl1789

LPP: lpp 1788 NOC: Noc_(—)1891 AEH: Mlg_(—)0688 Mlg_(—)2706 HHA:Hhal_(—)1685 HCH: HCH_(—)05299 CSA: Csal_(—)0301 Csal_(—)3068

ABO: ABO_(—)0648(fadAx)

MMW: Mmwyl1_(—)0073 Mmwyl1_(—)3021 Mmwyl1_(—)3053 Mmwyl1_(—)3097Mmwyl1_(—)4182

AHA: AHA_(—)2143(atoB)CVI: CV_(—)2088(atoB) CV_(—)2790(phaA)RSO: RSc0276(atoB) RSc1632(phbA) RSc1637(bktB) RSc1761(RS02948)

REU: Reut_A0138 Reut_A1348 Reut_A1353 Reut_B4561 Reut_B4738

-   -   Reut_B5587 Reut_C5943 Reut_C6062

REH: H16—A0170 H16—A0867 H16—A0868 H16—A0872 H16—A1297

-   -   H16—A1438(phaA) H16—A1445(bktB) H16—A1528 H16—A1713 H16—A1720    -   H16—A1887 H16—A2148 H16—B0380 H16—B0381 H16—B0406 H16—B0662    -   H16—B0668 H16—B0759 H16—B1369 H16—B1771

RME: Rmet_(—)0106 Rmet_(—)1357 Rmet_(—)1362 Rmet_(—)5156

BMA: BMA1316 BMA1321(phbA) BMA1436BMV: BMASAVP1_A1805(bktB) BMASAVP1_A1810(phbA)BML: BMA10299—A0086(phbA) BMA10299—A0091BMN: BMA10247_(—)1076(bktB) BMA10247_(—)1081(phbA)

BXE: Bxe_A2273 Bxe_A2335 Bxe_A2342 Bxe_A4255 Bxe_B0377 Bxe_B0739

-   -   Bxe_CO₃₃₂ Bxe_CO₅₇₄ Bxe_C0915

BVI: Bcep1808_(—)0519 Bcep1808_(—)1717 Bcep1808_(—)2877 Bcep1808_(—)3594

-   -   Bcep1808_(—)4015 Bcep1808_(—)5507 Bcep1808_(—)5644

BUR: Bcep18194_A3629 Bcep18194_A5080 Bcep18194_A5091

-   -   Bcep18194_A6102 Bcep18194_B0263 Bcep18194_B1439    -   Bcep18194_C6652 Bcep18194_C6802 Bcep18194_C6874    -   Bcep18194_C7118 Bcep18194_C7151 Bcep18194_C7332

BCN: Bcen_(—)1553 Bcen_(—)1599 Bcen_(—)2158 Bcen_(—)2563 Bcen_(—)2998Bcen_(—)6289 BCH: Bcen2424_(—)0542 Bcen2424_(—)1790 Bcen2424_(—)2772Bcen2424_(—)5368

-   -   Bcen2424_(—)6232 Bcen2424_(—)6276

BAM: Bamb_(—)0447 Bamb_(—)1728 Bamb_(—)2824 Bamb_(—)4717 Bamb_(—)5771Bamb_(—)5969

BPS: BPSL1426 BPSL1535(phbA) BPSL1540BPM: BURPS1710b_(—)2325(bktB) BURPS1710b_(—)2330(phbA)

-   -   BURPS1710b_(—)2453(atoB-2)        BPL: BURPS1106A_(—)2197(bktB) BURPS1106A_(—)2202(phbA)        BPD: BURPS668_(—)2160(bktB) BURPS668_(—)2165(phbA)

BTE: BTH_(—)12144 BTH_(—)12256 BTH_(—)12261 PNU: Pnuc_(—)0927 BPE:BP0447 BP0668 BP2059 BPA: BPP0608 BPP1744 BPP3805 BPP4216 BPP4361 BBR:BB0614 BB3364 BB4250 BB4804 BB4947 RFR: Rfer_(—)0272 Rfer_(—)1000Rfer_(—)1871 Rfer_(—)2273 Rfer_(—)2561 Rfer_(—)2594

-   -   Rfer_(—)3839

POL: Bpro_(—)1577 Bpro_(—)2140 Bpro_(—)3113 Bpro_(—)4187 PNA:Pnap_(—)0060 Pnap_(—)0458 Pnap_(—)0867 Pnap_(—)1159 Pnap_(—)2136Pnap_(—)2804 AAV: Aave_(—)0031 Aave_(—)2478 Aave_(—)3944 Aave_(—)4368AJS: Ajs_(—)0014 Ajs_(—)0124 Ajs_(—)1931 Ajs_(—)2073 Ajs_(—)2317Ajs_(—)3548

-   -   Ajs_(—)3738 Ajs_(—)3776

VEI: Veis_(—)1331 Veis_(—)3818 Veis_(—)4193 DAC: Daci_(—)0025Daci_(—)0192 Daci_(—)3601 Daci_(—)5988 MPT: Mpe_A1536 Mpe_A1776Mpe_A1869 Mpe_A3367

HAR: HEAR0577(phbA)MMS: mma_(—)0555NEU: NE2262(bktB)

NET: Neut_(—)0610

EBA: ebA5202 p2A409(tioL)AZO: azo0464(fadA1) azo0469(fadA2) azo2172(thlA)

DAR: Daro_(—)0098 Daro_(—)3022 HPA: HPAG1_(—)0675

HAC: Hac_(—)0958(atoB)

GME: Gmet_(—)1719 Gmet_(—)2074 Gmet_(—)2213 Gmet_(—)2268 Gmet_(—)3302GUR: Gura_(—)3043

BBA: Bd0404(atoB) Bd2095

DOL: Dole_(—)0671 Dole_(—)1778 Dole_(—)2160 Dole_(—)2187 ADE:Adeh_(—)0062 Adeh_(—)2365 AFW: Anae109_(—)0064 Anae109_(—)1504 MXA:MXAN_(—)3791 SAT: SYN_(—)02642 SFU: Sfum_(—)2280 Sfum_(—)3582 RPR: RP737RCO: RC1134 RC1135

RFE: RF_(—)0163(paaJ)RBE: RBE_(—)0139(paaJ)

RAK: A1C 05820 RBO: A1I_(—)07215 RCM: A1E_(—)04760

PUB: SAR11_(—)0428(thlA)MLO: mlr3847

MES: Meso_(—)3374 PLA: Plav_(—)1573 Plav_(—)2783

SME: SMa1450 SMc03879(phbA)

SMD: Smed_(—)0499 Smed_(—)3117 Smed_(—)5094 Smed_(—)5096

ATU: Atu2769(atoB) Atu3475ATC: AGR_C_(—)5022(phbA) AGR_L_(—)2713RET: RHE_CH04018(phbAch) RHE_PC00068(ypc00040) RHE_PF00014(phbAf)RLE: RL4621(phaA) pRL100301 pRL120369

BME: BMEIO274 BMEII0817

BMF: BAB1_(—)1783(phbA-1) BAB2_(—)0790(phbA-2)BMS: BR1772(phbA-1) BRA0448(phbA-2)BMB: BruAb1_(—)1756(phbA-1) BruAb2_(—)0774(phbA-2)BOV: BOV_(—)1707(phbA-1)

OAN: Oant_(—)1130 Oant_(—)3107 Oant_(—)3718 Oant_(—)4020

BJA: b110226(atoB) b113949 b117400 b117819 b1r3724(phbA)BRA: BRAD00562(phbA) BRAD00983(pimB) BRAD03110 BRADO3134(atoB)BBT: BBta_(—)3558 BBta_(—)3575(atoB) BBta_(—)5147(pimB)BBta_(—)7072(pimB)

-   -   BBta_(—)7614(phbA)        RPA: RPA0513(pcaF) RPA0531 RPA3715(pimB)

RPB: RPB_(—)0509 RPB_(—)0525 RPB_(—)1748 RPC: RPC_(—)0504 RPC_(—)0636RPC_(—)0641 RPC_(—)0832 RPC_(—)1050 RPC_(—)2005

-   -   RPC_(—)2194 RPC_(—)2228

RPD: RPD_(—)0306 RPD_(—)0320 RPD 3105 RPD_(—)3306 RPE: RPE 0168RPE_(—)0248 RPE_(—)3827 NWI: Nwi_(—)3060 XAU: Xaut_(—)3108 Xaut_(—)4665CCR: CC 0510 CC_(—)0894 CC_(—)3462

SIL: SP00142(bktB) SP00326(phbA) SP00773 SPO3408

SIT: TM1040_(—)0067 TM1040_(—)2790 TM1040_(—)3026 TM1040_(—)3735 RSP:RSP_(—)0745 RSP_(—)1354 RSP 3184 RSH: Rsph17029_(—)0022Rsph17029_(—)2401 Rsph17029_(—)3179 Rsph17029_(—)3921 RSQ:Rsph17025_(—)0012 Rsph17025_(—)2466 Rsph17025_(—)2833 JAN: Jann_(—)0262Jann_(—)0493 Jann_(—)4050

RDE: RD1_(—)0025 RD1_(—)0201(bktB) RD1_(—)3394(phbA)

PDE: Pden_(—)2026 Pden_(—)2663 Pden_(—)2870 Pden_(—)2907 Pden_(—)4811Pden_(—)5022 DSH: Dshi_(—)0074 Dshi_(—)3066 Dshi_(—)3331 MMR:Mmar10_(—)0697 HNE: HNE_(—)2706 HNE_(—)3065 HNE 3133 NAR: Saro_(—)0809Saro_(—)1069 Saro_(—)1222 Saro_(—)2306 Saro_(—)2349 SAL: Sala_(—)0781Sala_(—)1244 Sala_(—)2896 Sala 3158 SWI: Swit_(—)0632 Swit_(—)0752Swit_(—)2893 Swit_(—)3602 Swit_(—)4887 Swit_(—)5019

-   -   Swit_(—)5309

ELI: ELI 01475 ELI 06705 ELI 12035 GBE: GbCGDNIH1_(—)0447 ACR:Acry_(—)1847 Acry_(—)2256 RRU: Rru_A0274 Rru_A1380 Rru_A1469 Rru_A1946Rru_A3387

MAG: amb0842

MGM: Mmc1_(—)1165 ABA: Acid345_(—)3239

BSU: BG11319(mmgA) BG13063(yhfS)BHA: BH1997 BH2029 BH3801(mmgA)

BAN: BA3687 BA4240 BA5589 BAR: GBAA3687 GBAA4240 GBAA5589 BAA:BA_(—)0445 BA_(—)4172 BA_(—)4700 BAT: BAS3418 BAS3932 BAS5193 BCE:BC3627 BC4023 BC5344 BCA: BCE_(—)3646 BCE_(—)4076 BCE_(—)5475

BCZ: BCZK3329(mmgA) BCZK3780(thl) BCZK5044(atoB)

BCY: Bcer98_(—)2722 Bcer98_(—)3865

BTK: BT9727_(—)3379(mmgA) BT9727_(—)3765(thl) BT9727_(—)5028(atoB)BTL: BALH_(—)3262(mmgA) BALH_(—)3642(fadA) BALH_(—)4843(atoB)BLI: BL03925(mmgA)BLD: BLiO3968(mmgA)BCL: ABC0345 ABC2989 ABC3617 ABC3891(mmgA)

BAY: RBAM_(—)022450

BPU: BPUM_(—)2374(yhfS) BPUM_(—)2941 BPUM_(—)3373

OIH: OB06760B06890B26320B3013 GKA: GK1658 GK3397

SAU: SA0342 SA0534(vraB)SAV: SAV0354 SAV0576(vraB)SAM: MWO330 MWO531(vraB)SAR: SAR0351(thl) SAR0581

SAS: SAS0330 SAS0534

SAC: SACOL0426 SACOL0622(atoB)SAB: SAB0304(thl) SAB0526SAA: SAUSA300_(—)0355 SAUSA300_(—)0560(vraB)

SAO: SAOUHSC_(—)00336 SAOUHSC_(—)00558 SAJ: SaurJH9_(—)0402 SAH:SaurJH1_(—)0412 SEP: SE0346 SE2384 SER: SERP0032 SERP0220

SHA: SH0510(mvaC) SH2417

SSP: SSP0325 SSP2145

LMO: lmo1414LMF: LM0f2365_(—)1433LIN: lin1453LWE: lwe1431LLA: L11745(thiL) L25946(fadA)

LLC: LACR_(—)1665 LACR_(—)1956

LLM: llmg_(—)0930(thiL)SPY: SPy_(—)0140 SPy_(—)1637(atoB)SPZ: M5005—Spy_(—)0119 M5005—Spy_(—)0432 M5005—Spy_(—)1344(atoB)SPM: spyM18_(—)0136 spyM18_(—)1645(atoB)SPG: SpyM3_(—)0108 SpyM3_(—)1378(atoB)

SPS: SPs0110 SPs0484

SPH: MGAS10270—Spy0121 MGAS10270—Spy0433 MGAS10270—Spy1461(atoB)SPI: MGAS10750—Spy0124 MGAS10750—Spy0452 MGAS10750—Spy1453(atoB)SPJ: MGAS2096—Spy0123 MGAS2096—Spy0451 MGAS2096—Spy1365(atoB)SPK: MGAS9429—Spy0121 MGAS9429—Spy0431 MGAS9429—Spy1339(atoB)SPF: SpyM50447(atoB2)

SPA: M6—Spy0166 M6—Spy0466 M6—Spy1390

SPB: M28—Spy0117 M28—Spy0420 M28—Spy1385(atoB)

SAK: SAK_(—)0568 LJO: 111609

LAC: LBA0626(thiL)

LSA: LSA1486 LDB: Ldb0879 LBU: LBUL_(—)0804 LBR: LVIS_(—)2218 LCA:LSEI_(—)1787 LGA: LGAS_(—)1374 LRE: Lreu_(—)0052 EFA: EF1364 OOE:OEOE_(—)0529 STH: STH2913 STH725 STH804

CAC: CAC2873 CA_P0078(thiL)CPE: CPE2195(atoB)

CPF: CPF_(—)2460 CPR: CPR_(—)2170 CTC: CTC00312 CNO: NTO1CX_(—)0538NTO1CX_(—)0603

CDF: CD1059(th1A1) CD2676(th1A2)CBO: CBO3200(thl)

CBE: Cbei_(—)0411 Cbei_(—)3630

CKL: CKL_(—)3696(th1A1) CKL_(—)3697(th1A2) CKL_(—)3698(th1A3)

AMT: Amet_(—)4630 AOE: Clos_(—)0084 Clos_(—)0258

CHY: CHY_(—)1288 CHY_(—)1355(atoB) CHY_(—)1604 CHY_(—)1738

DSY: DSY0632 DSY0639 DSY1567 DSY1710 DSY2402 DSY3302 DRM: Dred_(—)0400Dred_(—)1491 Dred_(—)1784 Dred_(—)1892 SWO: Swol_(—)0308 Swol_(—)0675Swol_(—)0789 Swol_(—)1486 Swol_(—)1934 Swol_(—)2051

TTE: TTE0549(paaJ)

MTA: Moth_(—)1260

MTU: Rv1135A Rv1323(fadA4) Rv3546(fadA5)MTC: MT1365(phbA)MBO: Mb1167 Mb1358(fadA4) Mb3576(fadA5) Mb3586c(fadA6)MBB: BCG_(—)1197 BCG_(—)1385(fadA4) BCG_(—)3610(fadA5)BCG_(—)3620c(fadA6)MLE: ML1158(fadA4)MPA: MAP2407c(fadA3) MAP2436c(fadA4)

MAV: MAV_(—)1544 MAV_(—)1573 MAV_(—)1863 MAV_(—)5081 MSM: MSMEG_(—)2224MSMEG_(—)4920 MUL: MUL_(—)0357 MVA: Mvan_(—)1976 Mvan_(—)1988Mvan_(—)4305 Mvan_(—)4677 Mvan_(—)4891 MGI: Mflv_(—)1347 Mflv_(—)1484Mflv_(—)2040 Mflv_(—)2340 Mflv_(—)4356 Mfly_(—)4368 MMC: Mmcs_(—)1758Mmcs_(—)1769 Mmcs_(—)3796 Mmcs_(—)3864 MKM: Mkms_(—)0251 Mkms_(—)1540Mkms_(—)1805 Mkms_(—)1816 Mkms_(—)2836 Mkms_(—)3159

-   -   Mkms_(—)3286 Mkms_(—)3869 Mkms_(—)3938 Mkms_(—)4227 Mkms_(—)4411        Mkms_(—)4580    -   Mkms_(—)4724 Mkms_(—)4764 Mkms_(—)4776

MJL: Mjls_(—)0231 Mjls_(—)1739 Mjls_(—)1750 Mjls_(—)2819 Mjls_(—)3119Mjls_(—)3235

-   -   Mjls_(—)3800 Mjls_(—)3850 Mjls_(—)4110 Mjls_(—)4383 Mjls_(—)4705        Mjls_(—)4876    -   Mjls_(—)5018 Mjls_(—)5063 Mjls_(—)5075        CGL: NCg12309(cg12392)        CGB: cg2625(pcaF)

CEF: CE0731 CE2295

CJK: jk1543(fadA3)NFA: nfa10750(fadA4)RHA: RHA1_ro01455 RHA1_ro01623 RHA1_ro01876 RHA1_ro02517(catF)

-   -   RHA1_ro03022 RHA1_ro03024 RHA1_ro03391 RHA1_ro03892    -   RHA1_ro04599 RHA1_ro05257 RHA1_ro08871

SCO: SCO5399(SC8F4.03)

SMA: SAV1384(fadA5) SAV2856(fadA1)

ART: Arth_(—)1160 Arth_(—)2986 Arth_(—)3268 Arth_(—)4073 NCA:Noca_(—)1371 Noca_(—)1797 Noca_(—)1828 Noca_(—)2764 Noca_(—)4142 TFU:Tfu_(—)1520 Tfu_(—)2394 FRA: Francci3_(—)3687 FRE: Franean1_(—)1044Franean1_(—)2711 Franean1_(—)2726 Franean1_(—)3929

-   -   Franean1_(—)4037 Franean1_(—)4577        FAL: FRAAL2514 FRAAL2618 FRAAL5910(atoB)

ACE: Acel_(—)0626 Acel_(—)0672

SEN: SACE_(—)1192(mmgA) SACE_(—)2736(fadA6) SACE_(—)4011(catF)

-   -   SACE_(—)6236(fadA4)

STP: Strop_(—)3610 SAQ: Sare_(—)1316 Sare_(—)3991 RXY: Rxyl_(—)1582Rxyl_(—)1842 Rxyl_(—)2389 Rxyl_(—)2530 FNU: FN0495

BGA: BG0110(fadA)BAF: BAPKO_(—)0110(fadA)LIL: LA0457(thiL1) LA0828(thiL2) LA4139(fadA)LIC: LIC10396(phbA)LBJ: LBJ_(—)2862(paaJ-4)LBL: LBL_(—)0209(paaJ-4)SYN: s1r1993(phaA)SRU: SRU_(—)1211(atoB) SRU_(—)1547CHU: CHU_(—)1910(atoB)GFO: GFO_(—)1507(atoB)

FJO: Fjoh_(—)4612 FPS: FP0770 FP1586 FP1725 RRS: RoseRS_(—)3911RoseRS_(—)4348 RCA: Rcas_(—)0702 Rcas_(—)3206 HAU: Haur_(—)0522 DRA:DR_(—)1072 DR_(—)1428 DR_(—)1960 DR_(—)2480 DR_A0053 DGE: Dgeo_(—)0755Dgeo_(—)1305 Dgeo_(—)1441 Dgeo_(—)1883 TTH: TTC0191 TTCO0330 TTJ:TTHA0559 TME: Tmel_(—)1134 FNO: Fnod_(—)0314 PMO: Pmob_(—)0515

HMA: rrnAC0896(acaB3) rrnAC2815(aca2) rrnAC3497(yqeF)

-   -   rrnB0240(aca1) rrnB0242(acaB2) rrnB0309(acaB1)

TAC: Ta0582 TVO: TVN0649 PTO: PT01505 APE: APE 2108

SSO: SSO2377(acaB-4)

STO: ST0514

SAI: SacI_(—)0963 SacI_(—)1361(acaB1)

MSE: Msed_(—)0656 PAI: PAE1220 PIS: Pisl_(—)0029 Pisl_(—)1301 PCL:Pcal_(—)0781 PAS: Pars_(—)0309 Pars_(—)1071 CMA: Cmaq_(—)1941 ExemplaryHMG-CoA Synthase Nucleic Acids and Polypeptides HSA: 3157(HMGCS1)3158(HMGCS2) PTR: 457169(HMGCS2) 461892(HMGCS1) MCC: 702553(HMGCS1)713541(HMGCS2) MMU: 15360(Hmgcs2) 208715(Hmgcs1) RNO: 24450(Hmgcs2)29637(Hmgcs1) CFA: 479344(HMGCS 1) 607923(HMGCS2) BTA: 407767(HMGCS 1)SSC: 397673(CH242-38B5.1) GGA: 396379(HMGCS1)

XLA: 380091(hmgcsl) 447204(MGC80816)DRE: 394060(hmgcs1)

SPU: 578259(L00578259) DME: Dmel_CG4311(Hmgs) CEL: F25B4.6 ATH:AT4G11820(BAP1) OSA: 4331418 4347614 CME: CMM189C SCE: YML126C(ERG13)AGO: AGOS_ADL356C PIC: PICST_(—)83020 CAL: Ca019_(—)7312(Ca019.7312)CGR: CAGL0H04081 g

SPO: SPAC4F8.14c(hcs)

MGR: MGG_(—)01026 ANI: AN4923.2 AFM: AFUA_(—)3G10660 AFUA_(—)8G07210AOR: AO090003000611 AO090010000487 CNE: CNC05080 CNG02670 UMA: UM05362.1ECU: ECU10_(—)0510

DDI: DDBDRAFT_(—)0217522 DDB_(—)0219924(hgsA)

TET: TTHERM_(—)00691190 TBR: Tb927.8.6110 YPE: YP01457

YPK: y2712(pksG)YPM: YP_(—)1349(pksG)

YPA: YPA_(—)0750 YPN: YPN_(—)2521 YPP: YPDSF_(—)1517 YPS: YPTB1475 CBD:COXBU7E912_(—)1931 TCX: Tcr_(—)1719 DNO: DNO_(—)0799 BMA: BMAAl212 BPS:BPSS1002

BPM: BURPS1710b_A2613

BPL: BURPS1106A_A1384 BPD: BURPS668—A1470 BTE: BTH_II1670

MXA: MXAN_(—)3948(tac) MXAN_(—)4267(mvaS)BSU: BG10926(pksG)

OIH: OB2248

SAU: SA2334(mvaS)SAV: SAV2546(mvaS)SAM: MW2467(mvaS)SAR: SAR2626(mvaS)

SAS: SAS2432 SAC: SACOL2561

SAB: SAB2420(mvaS)

SAA: SAUSA300_(—)2484 SAO: SAOUHSC_(—)02860 SAJ: SaurJH9_(—)2569 SAH:SaurJH1_(—)2622 SEP: SE2110 SER: SERP2122

SHA: SH0508(mvaS)

SSP: SSPO₃₂₄

LMO: lmo1415LMF: LMOf2365_(—)1434(mvaS)LIN: lin1454LWE: lwel432(mvaS)LLA: L13187(hmcM)

LLC: LACR_(—)1666

LLM: llmg_(—)0929(hmcM)SPY: SPy_(—)0881(mvaS.2)SPZ: M5005—Spy_(—)0687(mvaS.1)SPM: spyM18_(—)0942(mvaS2)SPG: SpyM3_(—)0600(mvaS.2)

SPS: SPs1253

SPH: MGAS10270—Spy0745(mvaS1)SPI: MGAS 10750_Spy0779(mvaS 1)SPJ: MGAS2096—Spy0759(mvaS1)SPK: MGAS9429—Spy0743(mvaS1)SPF: SpyM51121(mvaS)

SPA: M6—Spy0704

SPB: M28—Spy0667(mvaS.1)

SPN: SP_(—)1727

SPR: spr1571(mvaS)SPD: SPD_(—)1537(mvaS)

SAG: SAG1316

SAN: gbs1386

SAK: SAK_(—)1347 SMU: SMU.943c

STC: str0577(mvaS)STL: stu0577(mvaS)

STE: STER_(—)0621

SSA: SSA_(—)0338(mvaS)

SSU: SSU05_(—)1641 SSV: SSU98_(—)1652 SGO: SGO 0244

LPL: lp_(—)2067(mvaS)

LJO: LJ1607

LAC: LBA0628(hmcS)LSA: LSA1484(mvaS)

LSL: LSL_(—)0526

LDB: Ldb0881(mvaS)

LBU: LBUL_(—)0806 LBR: LVIS_(—)1363 LCA: LSEI_(—)1785 LGA: LGAS_(—)1372LRE: Lreu_(—)0676 PPE: PEPE_(—)0868 EFA: EF1363 OOE: OEOE_(—)0968 LME:LEUM_(—)1184

NFA: nfa22120SEN: SACE_(—)4570(pksG)

BBU: BB0683 BGA: BG0706 BAF: BAPKO_(—)0727 FJO: Fjoh_(—)0678 HAL:VNG1615G(mvaB)

HMA: rrnAC1740(mvaS)

HWA: HQ2868A(mvaB) NPH: NP2608A(mvaB_(—)1) NP4836A(mvaB_(—)2) ExemplaryHydroxymethylglutaryl-CoA Reductase Nucleic Acids and Polypeptides HSA:3156(HMGCR) PTR: 471516(HMGCR) MCC: 705479(HMGCR) MMU: 15357(Hmgcr) RNO:25675(Hmgcr) CFA: 479182(HMGCR) BTA: 407159(HMGCR)

GGA: 395145(RCJMB04_(—)14 m24)

SPU: 373355(LOC373355) DME: Dmel_CG 10367 (Hmgcr) CEL: F08F8.2 OSA:4347443 SCE: YLR450W(HMG2) YML075C(HMG1) AGO: AGOS_AER152W CGR:CAGLOL11506 g

SPO: SPCC162.09c(hmgl)

ANI: AN3817.2 AFM: AFUA_(—)1G11230 AFUA_(—)2G03700 AOR: AO090103000311AO090120000217 CNE: CNF04830 UMA: UM03014.1 ECU: ECU10_(—)1720

DDI: DDB_(—)0191125(hmgA) DDB_(—)0215357(hmgB)

TBR: Tb927.6.4540 TCR: 506831.40 509167.20 LMA: LmjF30.3190 VCH: VCA0723VCO: VC0395_(—)0662 VVU: VV2_(—)0117 VVY: VVA0625 VPA: VPA0968 VFI:VFA0841 PAT: Patl_(—)0427 CBU: CBU_(—)0030 CBU_(—)0610

CBD: COXBU7E912_(—)0151 COXBU7E912_(—)0622(hmgA)

TCX: Tcr_(—)1717 DNO: DNO 0797 CVI: CV_(—)1806 SUS: Acid_(—)5728Acid_(—)6132

SAU: SA2333(mvaA)SAV: SAV2545(mvaA)SAM: MW2466(mvaA)SAB: SAB2419c(mvaA)

SEP: SE2109

LWE: lwe0819(mvaA)LLA: L10433(mvaA)

LLC: LACR_(—)1664

LLM: llmg_(—)0931(mvaA)SPY: SPy_(—)0880(mvaS.1)SPM: spyM18_(—)0941(mvaS1)SPG: SpyM3_(—)0599(mvaS.1)

SPS: SPs1254 SPH: MGAS10270—Spy0744 SPI: MGAS10750—Spy0778 SPJ:MGAS2096—Spy0758 SPK: MGAS9429—Spy0742 SPA: M6—Spy0703 SPN: SP_(—)1726SAG: SAG1317

SAN: gbs1387STC: str0576(mvaA)STL: stu0576(mvaA)

STE: STER_(—)0620

SSA: SSA_(—)0337(mvaA)LPL: lp_(—)0447(mvaA)

LJO: 111608 LSL: LSL_(—)0224 LBR: LVIS_(—)0450 LGA: LGAS_(—)1373 EFA:EF1364

NFA: nfa22110BGA: BG0708(mvaA)

SRU: SRU_(—)2422 FPS: FP2341

MMP: MMP0087(hmgA)

MMQ: MmarC5_(—)1589

MAC: MA3073(hmgA)

MBA: Mbar_A1972 MMA: MM_(—)0335 MBU: Mbur_(—)1098 MHU: Mhun_(—)3004 MEM:Memar_(—)2365 MBN: Mboo_(—)0137 MTH: MTH562

MST: Msp_(—)0584(hmgA)

MSI: Msm_(—)0227 MKA: MK0355(HMG1)

AFU: AF1736(mvaA)

HAL: VNG1875G(mvaA)

HMA: rrnAC3412(mvaA)

HWA: HQ3215A(hmgR) NPH: NP0368A(mvaA_(—)2) NP2422A(mvaA_(—)1) TAC:Ta0406m TVO: TVN1168 PTO: PT01143

PAB: PAB2106(mvaA)

PFU: PF1848 TKO: TK0914

RCI: RCIX1027(hmgA) RCIX376(hmgA)

APE: APE_(—)1869 IHO: Igni_(—)0476 HBU: Hbut_(—)1531 SSO: SSO0531 STO:ST1352 SAI: SacI_(—)1359 PAI: PAE2182 PIS: Pisl_(—)0814 PCL:Pcal_(—)1085 PAS: Pars_(—)0796 Exemplary Mevalonate Kinase Nucleic Acidsand Polypeptides HSA: 4598(MVK) MCC: 707645(MVK) MMU: 17855(Mvk) RNO:81727(Mvk) CFA: 486309(MVK) BTA: 505792(MVK) GGA: 768555(MVK) DRE:492477(zgc:103473) SPU: 585785(L00585785) DME: Dmel_CG33671 OSA: 4348331SCE: YMR208W(ERG12) AGO: AGOS_AER335W PIC: PICST_(—)40742(ERG12) CGR:CAGL0F03861 g SPO: SPAC13G6.11c MGR: MGG_(—)06946 ANI: AN3869.2 AFM:AFUA_(—)4G07780 AOR: AO090023000793 CNE: CNK01740 ECU: ECU09_(—)1780DDI: DDBDRAFT_(—)0168621 TET: TTHERM_(—)00637680 TBR: Tb927.4.4070 TCR:436521.9 509237.10 LMA: LmjF31.0560 CBU: CBU_(—)0608 CBU_(—)0609

CBD: COXBU7E912_(—)0620(mvk)LPN: lpg2039LPF: lpl2017

LPP: lpp 2022

BBA: Bd1027(1 mbP) Bd1630(mvk)MXA: MXAN_(—)5019(mvk)

OIH: OB0225

SAU: SA0547(mvaK1)SAV: SAV0590(mvaK1)SAM: MWO545(mvaK1)SAR: SAR0596(mvaK1)

SAS: SAS0549

SAC: SACOL0636(mvk)SAB: SAB0540(mvaK1)SAA: SAUSA300_(—)0572(mvk)

SAO: SAOUHSC_(—)00577 SEP: SE0361

SER: SERP0238(mvk)SHA: SH2402(mvaK1)

SSP: SSP2122

LMO: lmo0010LMF: LM0f2365_(—)0011LIN: lin0010LWE: lwe0011(mvk)LLA: L7866(yeaG)

LLC: LACR_(—)0454

LLM: llmg_(—)0425(mvk)SPY: SPy_(—)0876(mvaK1)SPZ: M5005—Spy_(—)0682(mvaK1)SPM: spyM18_(—)0937(mvaK1)SPG: SpyM3_(—)0595(mvaK1)

SPS: SPs1258

SPH: MGAS10270—Spy0740(mvaK1)SPI: MGAS10750—Spy0774(mvaK1)SPJ: MGAS2096—Spy0753(mvaK1)SPK: MGAS9429—Spy0737(mvaK1)SPF: SpyM51126(mvaK1)

SPA: M6—Spy0699

SPB: M28—Spy0662(mvaK1)

SPN: SP_(—)0381

SPR: spr0338(mvk)SPD: SPD_(—)0346(mvk)

SAG: SAG1326

SAN: gbs1396SAK: SAK_(—)1357(mvk)

SMU: SMU.181

STC: str0559(mvaK1)STL: stu0559(mvaK1)

STE: STER_(—)0598

SSA: SSA_(—)0333(mvaK1)

SSU: SSU05_(—)0289 SSV: SSU98_(—)0285

SGO: SGO_(—)0239(mvk)LPL: lp_(—)1735(mvaK1)

LJO: LJ1205

LAC: LBA1167(mvaK)LSA: LSA0908(mvaK1)LSL: LSL_(—)0685(eRG)LDB: Ldb0999(mvk)

LBU: LBUL_(—)0906 LBR: LVIS_(—)0858 LCA: LSEI_(—)1491 LGA: LGAS_(—)1033LRE: Lreu_(—)0915 PPE: PEPE_(—)0927

EFA: EF0904(mvk)

OOE: OEOE_(—)1100 LME: LEUM_(—)1385

NFA: nfa22070

BGA: BG0711 BAF: BAPKO_(—)0732 FPS: FP0313 MMP: MMP1335 MAE:Maeo_(—)0775

MAC: MA0602(mvk)

MBA: Mbar_A1421 MMA: MM_(—)1762 MBU: Mbur_(—)2395 MHU: Mhun_(—)2890 MEM:Memar_(—)1812 MBN: Mboo_(—)2213

MST: Msp_(—)0858(mvk)

MSI: Msm_(—)1439 MKA: MK0993(ERG12) HAL: VNG1145G(myk)

HMA: rrnAC0077(myk)

HWA: HQ2925A(mvk) NPH: NP2850A(mvk) PTO: PT01352 PHO: PH1625

PAB: PAB0372(mvk)PFU: PF1637(mvk)

TKO: TK1474

RCI: LRC399(mvk)

APE: APE_(—)2439 HBU: Hbut_(—)0877 SSO: SS00383 STO: ST2185

SAI: SacI_(—)2365(mvk)

MSE: Msed_(—)1602 PAI: PAE3108 PIS: Pisl_(—)0467 PCL: Pcal_(—)1835Exemplary Phosphomevalonate Kinase Nucleic Acids and Polypeptides HSA:10654(PMVK) PTR: 457350(PMVK) MCC: 717014(PMVK) MMU: 68603(Pmvk) CFA:612251(PMVK) BTA: 513533(PMVK) DME: Dmel_CG10268 ATH: AT1G31910 OSA:4332275 SCE: YMR220W(ERG8) AGO: AGOS_AER354W PIC: PICST_(—)52257(ERG8)CGR: CAGL0F03993 g SPO: SPAC343.01c MGR: MGG_(—)05812 ANI: AN2311.2 AFM:AFUA_(—)5G10680 AOR: AO090010000471 CNE: CNM00100 UMA: UM00760.1 DDI:DDBDRAFT_(—)0184512 TBR: Tb09.160.3690 TCR: 507913.20 508277.140 LMA:LmjF15.1460 MXA: MXAN_(—)5017 OIH: OB0227

SAU: SA0549(mvaK2)SAV: SAV0592(mvaK2)SAM: MWO547(mvaK2)SAR: SAR0598(mvaK2)

SAS: SAS0551 SAC: SACOL0638

SAB: SAB0542(mvaK2)

SAA: SAUSA300_(—)0574 SAO: SAOUHSC_(—)00579 SAJ: SaurJH9_(—)0615 SEP:SE0363 SER: SERP0240

SHA: SH2400(mvaK2)

SSP: SSP2120

LMO: lmo0012LMF: LM0f2365_(—)0013LIN: lin0012LWE: lwe0013LLA: L10014(yebA)

LLC: LACR_(—)0456

LLM: llmg_(—)0427SPY: SPy_(—)0878(mvaK2)SPZ: M5005—Spy_(—)0684(mvaK2)SPM: spyM18_(—)0939SPG: SpyM3_(—)0597(mvaK2)

SPS: SPs1256

SPH: MGAS 10270_Spy0742(mvaK2)SPI: MGAS 10750_Spy0776(mvaK2)SPJ: MGAS2096—Spy0755(mvaK2)SPK: MGAS9429—Spy0739(mvaK2)SPF: SpyM51124(mvaK2)

SPA: M6—Spy0701

SPB: M28—Spy0664(mvaK2)

SPN: SP_(—)0383

SPR: spr0340(mvaK2)SPD: SPD_(—)0348(mvaK2)

SAG: SAG1324

SAN: gbs1394

SAK: SAK_(—)1355 SMU: SMU.938

STC: str0561(mvaK2)STL: stu0561(mvaK2)

STE: STER_(—)0600

SSA: SSA_(—)0335(mvaK2)

SSU: SSU05_(—)0291 SSV: SSU98_(—)0287 SGO: SGO 0241

LPL: lp_(—)1733(mvaK2)

LJO: LJ1207 LAC: LBA1169

LSA: LSA0906(mvaK2)

LSL: LSL_(—)0683

LDB: Ldb0997(mvaK)

LBU: LBUL_(—)0904 LBR: LVIS_(—)0860 LCA: LSEI_(—)1092 LGA: LGAS_(—)1035LRE: Lreu_(—)0913 PPE: PEPE_(—)0925 EFA: EF0902

NFA: nfa22090

BGA: BG0710 BAF: BAPKO_(—)0731 NPH: NP2852A SSO: SSO2988 STO: ST0978SAI: Saci_(—)1244 Exemplary Diphosphomevalonate Decarboxylase NucleicAcids and Polypeptides HSA: 4597(MVD) PTR: 468069(MVD) MCC: 696865(MVD)MMU: 192156(Mvd) RNO: 81726(Mvd) CFA: 489663(MVD) GGA: 425359(MVD) DME:Dmel_CG8239 SCE: YNR043W(MVD1) AGO: AGOS_AGL232C PIC: PICST_(—)90752CGR: CAGL0C03630 g SPO: SPAC24C9.03 MGR: MGG_(—)09750 ANI: AN4414.2 AFM:AFUA_(—)4G07130 AOR: AO090023000862 CNE: CNL04950 UMA: UM05179.1 DDI:DDBDRAFT_(—)0218058 TET: TTHERM_(—)00849200 TBR: Tb10.05.0010Tb10.61.2745 TCR: 507993.330 511281.40 LMA: LmjF18.0020

CBU: CBU_(—)0607(mvaD)CBD: COXBU7E912_(—)0619(mvaD)LPN: lpg2040LPF: lpl2018LPP: lpp2023

TCX: Tcr_(—)1734

DNO: DNO_(—)0504(mvaD)

BBA: Bd1629

MXA: MXAN_(—)5018(mvaD)

OIH: OB0226

SAU: SA0548(mvaD)SAV: SAV0591(mvaD)SAM: MWO546(mvaD)SAR: SAR0597(mvaD)

SAS: SAS0550

SAC: SACOL0637(mvaD)SAB: SAB0541(mvaD)SAA: SAUSA300_(—)0573(mvaD)

SAO: SAOUHSC_(—)00578 SAJ: SaurJH9_(—)0614 SAH: SaurJH1_(—)0629 SEP:SE0362

SER: SERP0239(mvaD)SHA: SH2401(mvaD)

SSP: SSP2121

LMO: lmo0011LMF: LM0f2365_(—)0012(mvaD)LIN: lin0011LWE: lwe0012(mvaD)LLA: L9089(yeaH)

LLC: LACR_(—)0455

LLM: llmg_(—)0426(mvaD)SPY: SPy_(—)0877(mvaD)SPZ: M5005—Spy_(—)0683(mvaD)SPM: spyM18_(—)0938(mvd)SPG: SpyM3_(—)0596(mvaD)

SPS: SPs1257

SPH: MGAS10270—Spy0741(mvaD)SPI: MGAS 10750_Spy0775(mvaD)SPJ: MGAS2096—Spy0754(mvaD)SPK: MGAS9429—Spy0738(mvaD)SPF: SpyM51125(mvaD)

SPA: M6—Spy0700

SPB: M28—Spy0663(mvaD)

SPN: SP_(—)0382

SPR: spr0339(mvdl)SPD: SPD_(—)0347(mvaD)SAG: SAG1325(mvaD)SAN: gbs1395SAK: SAK_(—)1356(mvaD)

SMU: SMU.937

STC: str0560(mvaD)STL: stu0560(mvaD)

STE: STER_(—)0599

SSA: SSA_(—)0334(mvaD)

SSU: SSU05_(—)0290 SSV: SSU98_(—)0286

SGO: SGO_(—)0240(mvaD)LPL: 1p_(—)1734(mvaD)

LJO: LJ1206

LAC: LBA1168(mvaD)LSA: LSA0907(mvaD)

LSL: LSL_(—)0684

LDB: Ldb0998(mvaD)

LBU: LBUL_(—)0905 LBR: LVIS_(—)0859 LCA: LSEI_(—)1492 LGA: LGAS_(—)1034LRE: Lreu_(—)0914 PPE: PEPE_(—)0926

EFA: EF0903(mvaD)

LME: LEUM_(—)1386

NFA: nfa22080

BBU: BB0686 BGA: BG0709 BAF: BAPKO_(—)0730 GFO: GFO_(—)3632

FPS: FP0310(mvaD)

HAU: Haur_(—)1612 HAL: VNG0593G(dmd)

HMA: rrnAC1489(dmd)

HWA: HQ1525A(mvaD) NPH: NP1580A(mvaD) PTO: PT00478 PT01356 SSO: SSO2989STO: ST0977

SAI: Saci_(—)1245(mvd)

MSE: Msed_(—)1576 Exemplary Isopentenyl-Diphosphate Delta-Isomerase(IDI) Nucleic Acids and Polypeptides HSA: 3422(IDI1) 91734(ID12) PTR:450262(ID12) 450263(IDI1) MCC: 710052(LOC710052) 721730(LOC721730) MMU:319554(Idil) RNO: 89784(Idil) GGA: 420459(IDI1) XLA: 494671(LOC494671)

XTR: 496783(idi2)

SPU: 586184(L00586184)

CEL: K06H7.9(idi-1)

ATH: AT3G02780(IPP2) OSA: 4338791 4343523 CME: CMB062C SCE:YPL117C(IDI1) AGO: AGOS_ADL268C PIC: PICST_(—)68990(IDI1) CGR:CAGL0J06952 g

SPO: SPBC106.15(idi1)

ANI: AN0579.2 AFM: AFUA_(—)6G11160 AOR: AO090023000500 CNE: CNA02550UMA: UM04838.1 ECU: ECUO2_(—)0230

DDI: DDB_(—)0191342(ipi)

TET: TTHERM_(—)00237280 TTHERM_(—)00438860 TBR: Tb09.211.0700 TCR:408799.19 510431.10 LMA: LmjF35.5330

EHI: 46.t00025ECO: b2889(idi)ECJ: JW2857(idi)

ECE: Z4227 ECS: ECs3761

ECC: c3467

ECI: UTI89—C3274 ECP: ECP_(—)2882 ECV: APECO1_(—)3638

ECW: EcE24377A_(—)3215(idi)

ECX: EcHS_A3048 STY: STY3195

STT: t2957SPT: SPA2907(idi)SEC: SC2979(idi)STM: STM3039(idi)SFL: SF2875(idi)

SFX: S3074 SFV: SFV_(—)2937

SSN: SSON_(—)3042 SSON_(—)3489(yhfK)

SBO: SBO_(—)3103 SDY: SDY_(—)3193 ECA: ECA2789

PLU: p1u3987

ENT: Ent638_(—)3307 SPE: Spro_(—)2201 VPA: VPA0278 VFI: VF0403

PPR: PBPRA0469(mvaD)

PEN: PSEEN4850

CBU: CBU_(—)0607(mvaD)CBD: COXBU7E912_(—)0619(mvaD)LPN: lpg2051LPF: lpl2029

LPP: lpp 2034 TCX: Tcr_(—)1718 HHA: Hhal_(—)1623 DNO: DNO 0798

EBA: ebA5678 p2A143DVU: DVU1679(idi)

DDE: Dde_(—)1991 LIP: LI1134 BBA: Bd1626 AFW: Anae109_(—)4082

MXA: MXAN_(—)5021(fni)

RPR: RP452

RTY: RT0439(idi)

RCO: RC0744

RFE: RF_(—)0785(fni)RBE: RBE_(—)0731(fni)

RAK: A1C 04190 RBO: A1I_(—)04755 RCM: A1E_(—)02555 RRI: A1G_(—)04195

MLO: mlr6371RET: RHE_PD00245(ypd00046)

XAU: Xaut_(—)4134 SIL: SPO0131 SIT: TM1040_(—)3442 RSP: RSP_(—)0276 RSH:Rsph17029_(—)1919 RSQ: Rsph17025_(—)1019 JAN: Jann_(—)0168

RDE: RD1_(—)0147(idi)

DSH: Dshi_(—)3527

BSU: BG11440(ypgA)

BAN: BA1520 BAR: GBAA1520 BAA: BA_(—)2041 BAT: BAS 1409 BCE: BC1499 BCA:BCE_(—)1626

BCZ: BCZK1380(fni)

BCY: Bcer98_(—)1222

BTK: BT9727_(—)1381(fni)

BTL: BALH_(—)1354

BLI: BL02217(fni)

BLD: BLi02426

BAY: RBAM_(—)021020(fni)BPU: BPUM_(—)2020(fni)

OIH: OB0537

SAU: SA2136(fni)SAV: SAV2346(fni)SAM: MW2267(fni)SAR: SAR2431(fni)

SAS: SAS2237

SAC: SACOL2341(fni)SAB: SAB2225c(fni)SAA: SAUSA300_(—)2292(fni)

SAO: SAOUHSC_(—)02623 SEP: SE1925

SER: SERP1937(fni-2)SHA: SH0712(fni)

SSP: SSP0556 LMO: lmo 1383

LMF: LMOf2365_(—)1402(fni)LIN: lin1420LWE: lwe1399(fni)LLA: L11083(yebB)

LLC: LACR_(—)0457

LLM: llmg_(—)0428(fni)

SPY: SPy_(—)0879 SPZ: M5005—Spy_(—)0685

SPM: spyM18_(—)0940

SPG: SpyM3_(—)0598 SPS: SPs1255 SPH: MGAS10270—Spy0743 SPI:MGAS10750—Spy0777 SPJ: MGAS2096—Spy0756 SPK: MGAS9429—Spy0740

SPF: SpyM51123(fni)

SPA: M6—Spy0702 SPB: M28—Spy0665 SPN: SP_(—)0384

SPR: spr0341(fni)SPD: SPD_(—)0349(fni)

SAG: SAG1323

SAN: gbs1393SAK: SAK_(—)1354(fni)

SMU: SMU.939

STC: str0562(idi)STL: stu0562(idi)

STE: STER_(—)0601 SSA: SSA_(—)0336 SGO: SGO 0242

LPL: 1p_(—)1732(idi1)

LJO: LJ1208 LAC: LBA1171

LSA: LSA0905(idi)

LSL: LSL_(—)0682

LDB: Ldb0996(fni)

LBU: LBUL_(—)0903 LBR: LVIS_(—)0861 LCA: LSEI_(—)1493 LGA: LGAS_(—)1036LRE: Lreu_(—)0912 EFA: EF0901 00E: 0E0E_(—)1103 STH: STH1674 CBE:Cbei_(—)3081 DRM: Dred_(—)0474 SWO: Swol_(—)1341 MTA: Moth_(—)1328

MTU: Rv1745c(idi)MTC: MT1787(idi)MBO: Mb1774c(idi)MBB: BCG_(—)1784c(idi)

MPA: MAP3079c

MAV: MAV_(—)3894(fni)MSM: MSMEG_(—)1057(fni) MSMEG_(—)2337(fni)MUL: MUL_(—)0380(idi2)

MVA: Mvan_(—)1582 Mvan_(—)2176 MGI: Mflv_(—)1842 Mflv_(—)4187 MMC:Mmcs_(—)1954 MKM: Mkms_(—)2000 MJL: Mjls_(—)1934

CGL: NCg12223(cg12305)CGB: cg2531(idi)

CEF: CE2207

CDI: DIP1730(idi)NFA: nfa19790 nfa22100

RHA: RHA1_ro00239 SCO: SCO6750(SC5F2A.33c)

SMA: SAV1663(idi)LXX: Lxx23810(idi)CMI: CMM_(—)2889(idiA)AAU: AAur_(—)0321(idi)

PAC: PPA2115 FRA: Francci3_(—)4188 FRE: Franean15570

FAL: FRAAL6504(idi)

KRA: Krad_(—)3991

SEN: SACE_(—)2627(idiB_(—)2) SACE_(—)5210(idi)

STP: Strop_(—)4438 SAQ: Sare_(—)4564 Sare_(—)4928 RXY: Rxyl_(—)0400 BBU:BB0684 BGA: BG0707

SYN: sll1556SYC: syc2161_c

SYF: Synpcc7942_(—)1933

CYA: CYA_(—)2395(fni)CYB: CYB_(—)2691(fni)TEL: tll1403ANA: all4591

AVA: Ava_(—)2461 Ava_B0346 TER: Tery_(—)1589

SRU: SRU_(—)1900(idi)CHU: CHU_(—)0674(idi)GFO: GFO_(—)2363(idi)

FJO: Fjoh_(—)0269

FPS: FP1792(idi)

CTE: CT0257 CCH: Cag_(—)1445 CPH: Cpha266_(—)0385 PVI: Cvib_(—)1545 PLT:Plut_(—)1764 RRS: RoseRS_(—)2437 RCA: Rcas_(—)2215 HAU: Haur_(—)4687DRA: DR_(—)1087 DGE: Dgeo_(—)1381 TTH: TT_P0067 TTJ: TTHB 110 MJA:MJ0862 MMP: MMP0043 MMQ: MmarC5_(—)1637 MMX: MmarC6_(—)0906 MMZ:MmarC7_(—)1040 MAE: Maeo_(—)1184 MVN: Mevan_(—)1058

MAC: MA0604(idi)

MBA: Mbar_A1419 MMA: MM_(—)1764 MBU: Mbur_(—)2397 MTP: Mthe_(—)0474 MHU:Mhun_(—)2888 MLA: Mlab_(—)1665 MEM: Memar_(—)1814 MBN: Mboo_(—)2211 MTH:MTH48

MST: Msp_(—)0856(fni)

MSI: Msm_(—)1441 MKA: MK0776(11dD) AFU: AF2287 HAL: VNG1818G(idi)VNG6081G(crt_(—)1) VNG6445G(crt_(—)2) VNG7060 VNG7149

HMA: rrnAC3484(idi)

HWA: HQ2772A(idiA) HQ2847A(idiB) NPH: NP0360A(idiB_(—)1) NP4826A(idiA)NP5124A(idiB_(—)2) TAC: Ta0102 TVO: TVN0179 PTO: PTO0496 PHO: PH1202PAB: PAB1662 PFU: PF0856 TKO: TK1470

RCI: LRC397(fni)

APE: APE_(—)1765.1 SMR: Smar_(—)0822 IHO: Igni_(—)0804 HBU: Hbut_(—)0539SSO: SS00063 STO: ST2059 SAI: Saci_(—)0091 MSE: Msed_(—)2136 PAI:PAE0801 PIS: Pis1_(—)1093 PCL: Pcal_(—)0017 PAS: Pars 0051 TPE:Tpen_(—)0272 Exemplary Isoprene Synthase Nucleic Acids and PolypeptidesGenbank Accession Nos. AY341431 AY316691 AY279379 AJ457070

AY182241

1-29. (canceled)
 30. A method of producing isoprene, the methodcomprising: (a) culturing recombinant cells comprising a heterologousisoprene synthase polypeptide under suitable culture conditions for theproduction of isoprene in a gas phase; and (b) producing isoprene in thegas phase, wherein the gas phase comprises a nonflammable concentrationof isoprene and greater than or about 9.5% (volume) oxygen, and whereinthe cells produce greater than about 400 nmole/g_(wcm)/hr of isoprene.31. (canceled)
 32. The method of claim 30, wherein the concentration ofisoprene in the gas phase is less than the lower flammability limit(LFL) of isoprene.
 33. The method of claim 32, wherein the concentrationof isoprene in the gas phase is less than about 1.5 vol. %.
 34. Themethod of claim 30, wherein the concentration of isoprene in the gasphase is greater than the upper flammability limit (UFL) of isoprene.35. The method of claim 34, wherein the concentration of isoprene in thegas phase is greater than about 12.0 vol. %.
 36. The method of claim 30,wherein isoprene is produced in the gas phase at a temperature betweenabout 25° C. to about 55° C.
 37. The method of claim 30, whereinisoprene is produced in the gas phase at a pressure between about 1atmosphere and 3 atmospheres.
 38. The method of claim 30, wherein theisoprene synthase polypeptide is a plant isoprene synthase polypeptide.39. The method of claim 30, wherein the recombinant cells aregram-positive bacterial cells, gram-negative bacterial cells, fungalcells, filamentous fungal cells, algal cells or yeast cells.
 40. Themethod of claim 39, wherein the cells are gram-positive bacterial cellor gram-negative bacterial cells.
 41. The method of claim 40, whereinthe bacterial cells are selected from the group consisting ofEscherichia coli, Pantoae citrea, Bacillus subtilis, Bacilluslicheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus clausii, Bacillus halodurans, Bacillus megaterium, Bacilluscoagulans, Bacillus circulans, Bacillus lautus, Bacillus thuringiensis,Streptomyces lividans, Streptomyces coelicolor, Streptomyces griseus,Pseudomonas sp., and Pseudomonas alcaligenes cells.
 42. The method ofclaim 39, wherein the cells are fungal cells.
 43. The method of claim42, wherein the fungal cells are selected from the group consisting ofAspergillus sp., yeast, Trichoderma sp., Yarrowia sp., Saccharomycessp., Schizosaccharomyces sp., Pichia sp., Candida sp., Aspergillusoryzae, Aspergillus niger, Saccharomyces cerevisiae, Schizosaccharomycespombe, Trichoderma reesei, Humicola insolens, Humicola lanuginose,Humicola grisea, Candida lucknowense, Aspergillus sojae, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus aculeatus, Aspergillusawamori, Fusarium roseum, Fusarium graminum, Fusarium cerealis, Fusariumoxysporuim, F. venenatum, Neurospora crassa, Mucor miehei, Trichodermaviride, Fusarium oxysporum, and Fusarium solani cells.
 44. The method ofclaim 39, wherein the cells are algal cells.
 45. The method of claim 44,wherein the algal cells are selected from the group consisting of greenalgae, red algae, glaucophytes, chlorarachniophytes, euglenids,chromista, and dinoflagellates
 46. The method of claim 30, wherein thecells further comprise a nucleic acid encoding an isopentyl-diphosphatedelta-isomerase (IDI) polypeptide.
 47. The method of claim 46, whereinthe nucleic acid encoding the IDI polypeptide is a heterologous nucleicacid.
 48. The method of claim 46, wherein the IDI polypeptide is a yeastIDI polypeptide.
 49. The method of claim 46, wherein the nucleic acidencoding the IDI polypeptide is a copy of an endogenous nucleic acidencoding an IDI polypeptide.
 50. The method of claim 30, wherein thecells further comprise a heterologous nucleic acid encoding a1-deoxyxylulose-5-phosphate synthase (DXS) polypeptide.
 51. The methodof claim 30, wherein the cells further comprise a copy of an endogenousnucleic acid encoding a DXS polypeptide.
 52. The method of claim 30,wherein the cells further comprise one or more heterologous nucleicacids encoding one or more mevalonate (MVA) pathway polypeptides. 53.The method of claim 30, wherein the cells further comprise one or morecopies of endogenous nucleic acids encoding one or more MVA pathwaypolypeptides.
 54. The method of claim 30, wherein the cells furthercomprise one or more nucleic acids encoding the polypeptides of theentire MVA pathway.
 55. The method of claim 30, wherein the cellsfurther comprise: (a) a nucleic acid encoding an isopentyl-diphosphatedelta-isomerase (IDI) polypeptide; and (b) (i) one or more nucleic acidsencoding one or more non-mevalonate (DXP) pathway polypeptides and/or(ii) one or more nucleic acids encoding one or more mevalonate (MVA)pathway polypeptides.
 56. The method of claim 55, wherein the one ormore nucleic acids encoding one or more DXP pathway polypeptides areheterologous nucleic acids.
 57. The method of claim 55, wherein the oneor more nucleic acids encoding one or more DXP pathway polypeptides areendogenous nucleic acids.
 58. The method of claim 55, wherein said oneor more nucleic acids encoding one or more MVA pathway polypeptides areheterologous nucleic acids.
 59. The method of claim 55, wherein said oneor more nucleic acids encoding one or more MVA pathway polypeptides areendogenous nucleic acids.